2,2-difluoropropionamide derivatives of bardoxolone methyl, polymorphic forms and methods of use thereof

ABSTRACT

The present invention relates generally to the compound:
         N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide,
 
polymorphic forms thereof, methods for preparation and use thereof, pharmaceutical compositions thereof, and kits and articles of manufacture thereof.

The present application claims the benefit of priority to U.S.Provisional Application No. 61/780,444, filed on Mar. 13, 2013, U.S.Provisional Application No. 61/775,288, filed on Mar. 8, 2013, and U.S.Provisional Application No. 61/687,669, filed on Apr. 27, 2012; theentire contents of each are herein incorporated by reference.

Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewithas an ASCII compliant text file named “REATP0073US_ST25,” created onApr. 24, 2013 and having a size of ˜6 kilobytes. The content of theaforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the compound:

-   -   N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide,        also referred to herein as RTA 408, 63415, or PP415. The present        invention also relates to polymorphic forms thereof, methods for        preparation and use thereof, pharmaceutical compositions        thereof, and kits and articles of manufacture thereof.

II. Description of Related Art

The anti-inflammatory and anti-proliferative activity of the naturallyoccurring triterpenoid, oleanolic acid, has been improved by chemicalmodifications. For example,2-cyano-3,12-diooxooleana-1,9(11)-dien-28-oic acid (CDDO) and relatedcompounds have been developed. See Honda et al., 1997; Honda et al.,1998; Honda et al., 1999; Honda et al., 2000a; Honda et al., 2000b;Honda et al., 2002; Suh et al., 1998; Suh et al., 1999; Place et al.,2003; Liby et al., 2005; and U.S. Pat. Nos. 8,129,429, 7,915,402,8,124,799, and 7,943,778, all of which are incorporated herein byreference. The methyl ester, bardoxolone methyl (CDDO-Me), has beenevaluated in phase II and III clinical trials for the treatment andprevention of diabetic nephropathy and chronic kidney disease. SeePergola et al., 2011, which is incorporated herein by reference.

Synthetic triterpenoid analogs of oleanolic acid have also been shown tobe inhibitors of cellular inflammatory processes, such as the inductionby IFN-γ of inducible nitric oxide synthase (iNOS) and of COX-2 in mousemacrophages. See Honda et al, (2000a), Honda et al. (2000b), Honda etal. (2002), and U.S. Pat. Nos. 8,129,429, 7,915,402, 8,124,799, and7,943,778, which are all incorporated herein by reference. Compoundsderived from oleanolic acid have been shown to affect the function ofmultiple protein targets and thereby modulate the activity of severalimportant cellular signaling pathways related to oxidative stress, cellcycle control, and inflammation (e.g., Dinkova-Kostova et al., 2005;Ahmad et al., 2006; Ahmad et al., 2008; Liby et al., 2007a, and U.S.Pat. Nos. 8,129,429, 7,915,402, 8,124,799, and 7,943,778).

Given that the biological activity profiles of known triterpenoidderivatives vary, and in view of the wide variety of diseases that maybe treated or prevented with compounds having potent antioxidant andanti-inflammatory effects, and the high degree of unmet medical needrepresented within this variety of diseases, it is desirable tosynthesize new compounds with different biological activity profiles forthe treatment or prevention of one or more indications.

SUMMARY OF THE INVENTION

In some aspects of the present invention, there is provided a compoundof the formula (also referred to herein as RTA 408, 63415, or PP415):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is in the form of a pharmaceuticallyacceptable salt. In some embodiments, the compound is not in the form ofa salt.

In another aspect of the present invention, there are providedpolymorphic forms of the above compound. In some embodiments, thepolymorphic form has an X-ray powder diffraction pattern (CuKα)comprising a halo peak at about 14°2θ. In some embodiments, the X-raypowder diffraction pattern (CuKα) further comprises a shoulder peak atabout 8°2θ. In some embodiments, the X-ray powder diffraction pattern(CuKα) is substantially as shown in FIG. 59. In some embodiments, thepolymorphic form has a T_(g) from about 150° C. to about 155° C.,including for example, a T_(g) of about 153° C. or a T_(g) of about 150°C. In some embodiments, the polymorphic form has a differential scanningcalorimetry (DSC) curve comprising an endotherm centered from about 150°C. to about 155° C. In some embodiments, the endotherm is centered atabout 153° C. In some embodiments, the endotherm is centered at about150° C. In some embodiments, the differential scanning calorimetry (DSC)curve is substantially as shown in FIG. 62.

In some embodiments, the polymorphic form is a solvate having an X-raypowder diffraction pattern (CuKα) comprising peaks at about 5.6, 7.0,10.6, 12.7, and 14.6°2θ. In some embodiments, the X-ray powderdiffraction pattern (CuKα) is substantially as shown in FIG. 75, toppattern.

In some embodiments, the polymorphic form is a solvate having an X-raypowder diffraction pattern (CuKα) comprising peaks at about 7.0, 7.8,8.6, 11.9, 13.9 (double peak), 14.2, and 16.0°2θ. In some embodiments,the X-ray diffraction pattern (CuKα) is substantially as shown in FIG.75, second pattern from top.

In some embodiments, the polymorphic form is an acetonitrile hemisolvatehaving an X-ray powder diffraction pattern (CuKα) comprising peaks atabout 7.5, 11.4, 15.6 and 16.6°2θ. In some embodiments, the X-raydiffraction pattern (CuKα) is substantially as shown in FIG. 75, secondpattern from bottom. In some embodiments, the polymorphic form has aT_(g) of about 196° C. In some embodiments, the polymorphic form has adifferential scanning calorimetry (DSC) curve comprising an endothermcentered at about 196° C. In some embodiments, the differential scanningcalorimetry (DSC) curve is substantially as shown in FIG. 116.

In some embodiments, the polymorphic form is a solvate having an X-raypowder diffraction pattern (CuKα) comprising peaks at about 6.8, 9.3,9.5, 10.5, 13.6, and 15.6°2θ. In some embodiments, the X-ray diffractionpattern (CuKα) is substantially as shown in FIG. 75, bottom pattern.

In another aspect of the present invention, there are providedpharmaceutical compositions comprising an active ingredient consistingof the above compound or a polymorphic form thereof (such as, e.g., anyone of the polymorphic forms described herein above and below), and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition is formulated for administration: orally,intraadiposally, intraarterially, intraarticularly, intracranially,intradermally, intralesionally, intramuscularly, intranasally,intraocularly, intrapericardially, intraperitoneally, intrapleurally,intraprostatically, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, locally, mucosally,parenterally, rectally, subconjunctival, subcutaneously, sublingually,topically, transbuccally, transdermally, vaginally, in crèmes, in lipidcompositions, via a catheter, via a lavage, via continuous infusion, viainfusion, via inhalation, via injection, via local delivery, or vialocalized perfusion. In some embodiments, the pharmaceutical compositionis formulated for oral, intraarterial, intravenous, or topicaladministration. In some embodiments, the pharmaceutical composition isformulated for oral administration.

In some embodiments, the pharmaceutical composition is formulated as ahard or soft capsule, a tablet, a syrup, a suspension, an emulsion, asolution, a solid dispersion, a wafer, or an elixir. In someembodiments, the pharmaceutical composition according to the inventionfurther comprises an agent that enhances solubility and dispersibility.(For example, agents that enhance solubility and dispersibility include,but are not limited to, PEGs, cyclodextrans, and cellulose derivatives.)In some embodiments, the compound or polymorphic form is suspended insesame oil.

In other embodiments, the pharmaceutical composition is formulated fortopical administration. In other embodiments, the pharmaceuticalcomposition is formulated as a lotion, a cream, a gel, an oil, anointment, a salve, an emulsion, a solution, or a suspension. In someembodiments, the pharmaceutical composition is formulated as a lotion,as a cream, or as a gel. In some embodiments, the amount of the activeingredient is from about 0.01% to about 5% by weight, about 0.01% toabout 3% by weight, or 0.01%, 0.1%, 1%, or 3% by weight.

In another aspect of the present invention there are provided methods oftreating or preventing a condition associated with inflammation oroxidative stress in a patient in need thereof, comprising administeringto the patient a therapeutically effective amount of the pharmaceuticalcomposition as described above or below. The invention likewise relatesto the compound N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide(or RTA 408) or a pharmaceutically acceptable salt thereof, or apolymorphic form of that compound (such as, e.g., any one of thepolymorphic forms described herein above or below), or a pharmaceuticalcomposition comprising any of the aforementioned entities and apharmaceutically acceptable carrier (including, e.g., the pharmaceuticalcompositions described above), for use in treating or preventing acondition associated with inflammation or oxidative stress. Theinvention also relates to the use of the aforementioned compound,polymorphic form or pharmaceutical composition for the preparation of amedicament for the treatment or prevention of a condition associatedwith inflammation or oxidative stress. In some embodiments, thecondition is associated with inflammation. In other embodiments, thecondition is associated with oxidative stress. In some embodiments, thecondition is a skin disease or disorder, sepsis, dermatitis,osteoarthritis, cancer, inflammation, an autoimmune disease,inflammatory bowel disease, a complication from localized or total-bodyexposure to ionizing radiation, mucositis, acute or chronic organfailure, liver disease, pancreatitis, an eye disorder, a lung disease ordiabetes.

The present invention furthermore relates to the compoundN-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide(or RTA 408) or a pharmaceutically acceptable salt thereof, or apolymorphic form of that compound (such as, e.g., any one of thepolymorphic forms described herein above or below), or a pharmaceuticalcomposition comprising any of the aforementioned entities and apharmaceutically acceptable carrier (including, e.g., the pharmaceuticalcompositions described above), for use in treating or preventing acondition selected from a skin disease or disorder, sepsis, dermatitis,osteoarthritis, cancer, inflammation, an autoimmune disease,inflammatory bowel disease, a complication from localized or total-bodyexposure to ionizing radiation, mucositis, acute or chronic organfailure, liver disease, pancreatitis, an eye disorder, a lung disease,or diabetes. Accordingly, the invention relates to the use of theaforementioned compound, polymorphic form or pharmaceutical compositionfor the preparation of a medicament for the treatment or prevention of acondition selected from a skin disease or disorder, sepsis, dermatitis,osteoarthritis, cancer, inflammation, an autoimmune disease,inflammatory bowel disease, a complication from localized or total-bodyexposure to ionizing radiation, mucositis, acute or chronic organfailure, liver disease, pancreatitis, an eye disorder, a lung disease,or diabetes. The invention also relates to a method of treating orpreventing a condition selected from a skin disease or disorder, sepsis,dermatitis, osteoarthritis, cancer, inflammation, an autoimmune disease,inflammatory bowel disease, a complication from localized or total-bodyexposure to ionizing radiation, mucositis, acute or chronic organfailure, liver disease, pancreatitis, an eye disorder, a lung disease,or diabetes in a patient in need thereof, the method comprisingadministering to the patient a therapeutically effective amount of theaforementioned compound, polymorphic form or pharmaceutical composition.In some embodiments, the condition is a skin disease or disorder such asdermatitis, a thermal or chemical burn, a chronic wound, acne, alopecia,other disorders of the hair follicle, epidermolysis bullosa, sunburn,complications of sunburn, disorders of skin pigmentation, anaging-related skin condition; a post-surgical wound, a scar from a skininjury or burn, psoriasis, a dermatological manifestation of anautoimmune disease or a graft-versus host disease, skin cancer, or adisorder involving hyperproliferation of skin cells. In someembodiments, the skin disease or disorder is dermatitis. In someembodiments, the dermatitis is allergic dermatitis, atopic dermatitis,dermatitis due to chemical exposure, or radiation-induced dermatitis. Inother embodiments, the skin disease or disorder is a chronic wound. Insome embodiments, the chronic wound is a diabetic ulcer, a pressuresore, or a venous ulcer. In other embodiments, the skin disease ordisorder is alopecia. In some embodiments, the alopecia is selected frombaldness or drug-induced alopecia. In other embodiments, the skindisease or disorder is a disorder of skin pigmentation. In someembodiments, the disorder of skin pigmentation is vitiligo. In otherembodiments, the skin disease or disorder is a disorder involvinghyperproliferation of skin cells. In some embodiments, the disorderinvolving hyperproliferation of skin cells is hyperkeratosis.

In other embodiments, the condition is an autoimmune disease, such asrheumatoid arthritis, lupus, Crohn's disease, or psoriasis. In otherembodiments, the condition is a liver disease, such as fatty liverdisease or hepatitis.

In other embodiments, the condition is an eye disorder, such as uveitis,macular degeneration, glaucoma, diabetic macular edema, blepharitis,diabetic retinopathy, a disease or disorder of the corneal endothelium,post-surgical inflammation, dry eye, allergic conjunctivitis or a formof conjunctivitis. In some embodiments, the eye disorder is maculardegeneration. In some embodiments, the macular degeneration is the dryform. In other embodiments, the macular degeneration is the wet form. Insome embodiments, the disease or disorder of the corneal endothelium isFuchs endothelial corneal dystrophy.

In other embodiments, the condition is a lung disease, such as pulmonaryinflammation, pulmonary fibrosis, COPD, asthma, cystic fibrosis, oridiopathic pulmonary fibrosis. In some embodiments, the COPD is inducedby cigarette smoke.

In other embodiments, the condition is sepsis. In other embodiments, thecondition is mucositis resulting from radiation therapy or chemotherapy.In some embodiments, the mucositis presents orally. In otherembodiments, the condition is associated with exposure to radiation. Insome embodiments, the radiation exposure leads to dermatitis. In someembodiments, the radiation exposure is acute. In other embodiments, theradiation exposure is fractionated.

In other embodiments, the condition is cancer. In some embodiments, thecancer is a carcinoma, sarcoma, lymphoma, leukemia, melanoma,mesothelioma, multiple myeloma, or seminoma. In other embodiments, thecancer is of the bladder, blood, bone, brain, breast, central nervoussystem, cervix, colon, endometrium, esophagus, gall bladder, genitalia,genitourinary tract, head, kidney, larynx, liver, lung, muscle tissue,neck, oral or nasal mucosa, ovary, pancreas, prostate, skin, spleen,small intestine, large intestine, stomach, testicle, or thyroid.

In some embodiments, the pharmaceutical composition is administeredbefore or immediately after a subject is treated with a radiationtherapy or a chemotherapy wherein the chemotherapy does not comprise RTA408 or its polymorphic forms. In some embodiments, the pharmaceuticalcomposition is administered both before and after the subject is treatedwith radiation therapy, chemotherapy or both. In some embodiments, thetreatment reduces a side effect of the radiation therapy or thechemotherapy. In some embodiments, the side effect is mucositis anddermatitis. In some embodiments, the treatment enhances the efficacy ofthe radiation therapy or the chemotherapy. In some embodiments, thechemotherapy comprises administering to the patient a therapeuticallyeffective amount of 5-fluorouracil or docetaxel.

Additional combination treatment therapy is also contemplated by thepresent disclosure. For example, in some embodiments, the methods oftreating cancer in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure, the methods may further comprise one or more treatmentsselected from the group consisting of administering a pharmaceuticallyeffective amount of a second drug, radiotherapy, immunotherapy, genetherapy, and surgery. In some embodiments, the methods may furthercomprise (1) contacting a tumor cell with the compound prior tocontacting the tumor cell with the second drug, (2) contacting a tumorcell with the second drug prior to contacting the tumor cell with thecompound, or (3) contacting a tumor cell with the compound and thesecond drug at the same time. The second drug may, in certainembodiments, be an antibiotic, anti-inflammatory, anti-neoplastic,anti-proliferative, anti-viral, immunomodulatory, or immunosuppressive.In other embodiments, the second drug may be an alkylating agent,androgen receptor modulator, cytoskeletal disruptor, estrogen receptormodulator, histone-deacetylase inhibitor, HMG-CoA reductase inhibitor,prenyl-protein transferase inhibitor, retinoid receptor modulator,topoisomerase inhibitor, or tyrosine kinase inhibitor. In certainembodiments, the second drug is 5-azacitidine, 5-fluorouracil,9-cis-retinoic acid, actinomycin D, alitretinoin, all-trans-retinoicacid, annamycin, axitinib, belinostat, bevacizumab, bexarotene,bosutinib, busulfan, capecitabine, carboplatin, carmustine, CD437,cediranib, cetuximab, chlorambucil, cisplatin, cyclophosphamide,cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine, docetaxel,dolastatin-10, doxifluridine, doxorubicin, doxorubicin, epirubicin,erlotinib, etoposide, gefitinib, gemcitabine, gemtuzumab ozogamicin,hexamethylmelamine, idarubicin, ifosfamide, imatinib, irinotecan,isotretinoin, ixabepilone, lapatinib, LBH589, lomustine,mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin,mitoxantrone, MS-275, neratinib, nilotinib, nitrosourea, oxaliplatin,paclitaxel, plicamycin, procarbazine, semaxanib, semustine, sodiumbutyrate, sodium phenylacetate, streptozotocin, suberoylanilidehydroxamic acid, sunitinib, tamoxifen, teniposide, thiopeta, tioguanine,topotecan, TRAIL, trastuzumab, tretinoin, trichostatin A, valproic acid,valrubicin, vandetanib, vinblastine, vincristine, vindesine, orvinorelbine.

Methods of treating or preventing a disease with an inflammatorycomponent in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure are also contemplated. In some embodiments, the disease maybe, for example, lupus or rheumatoid arthritis. In other embodiments,the disease may be an inflammatory bowel disease, such as Crohn'sdisease or ulcerative colitis. In other embodiments, the disease with aninflammatory component may be a cardiovascular disease. In otherembodiments, the disease with an inflammatory component may be diabetes,such as type 1 or type 2 diabetes. In other embodiments, RTA 408, itspolymorphs, and pharmaceutical compositions may also be used to treatcomplications associated with diabetes. Such complications arewell-known to a person of skill in the art and include but are notlimited to, for example, obesity, hypertension, atherosclerosis,coronary heart disease, stroke, peripheral vascular disease,hypertension, nephropathy, neuropathy, myonecrosis, retinopathy andmetabolic syndrome (syndrome X). In other embodiments, the disease withan inflammatory component may be a skin disease, such as psoriasis,acne, or atopic dermatitis. Administration of RTA 408, its polymorphs,and pharmaceutical compositions in treatment methods of such skindiseases may be but are not limited to, for example, topical or oral.

In other embodiments, the disease with an inflammatory component may bemetabolic syndrome (syndrome X). A patient having this syndrome ischaracterized as having three or more symptoms selected from thefollowing group of five symptoms: (1) abdominal obesity; (2)hypertriglyceridemia; (3) low high-density lipoprotein cholesterol(HDL); (4) high blood pressure; and (5) elevated fasting glucose, whichmay be in the range characteristic of Type 2 diabetes if the patient isalso diabetic. Each of these symptoms is defined in the Third Report ofthe National Cholesterol Education Program Expert Panel on Detection,Evaluation and Treatment of High Blood Cholesterol in Adults (AdultTreatment Panel III, or ATP III), National Institutes of Health, 2001,NIH Publication No. 01-3670, which is incorporated herein by reference.Patients with metabolic syndrome, whether or not they have or developovert diabetes mellitus, have an increased risk of developing themacrovascular and microvascular complications that are listed above thatoccur with type 2 diabetes, such as atherosclerosis and coronary heartdisease.

Another general method of the present disclosure entails a method oftreating or preventing a cardiovascular disease in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure. In some embodiments, thecardiovascular disease may be but not limited to, for example,atherosclerosis, cardiomyopathy, congenital heart disease, congestiveheart failure, myocarditis, rheumatic heart disease, valve disease,coronary artery disease, endocarditis, or myocardial infarction.Combination therapy is also contemplated for methods of treating orpreventing a cardiovascular disease in a subject. For example, suchmethods may further comprise administering a pharmaceutically effectiveamount of one or more cardiovascular drugs. The cardiovascular drug maybe but not limited to, for example, a cholesterol lowering drug, ananti-hyperlipidemic, a calcium channel blocker, an anti-hypertensive, oran HMG-CoA reductase inhibitor. In some embodiments, non-limitingexamples of cardiovascular drugs include amlodipine, aspirin, ezetimibe,felodipine, lacidipine, lercanidipine, nicardipine, nifedipine,nimodipine, nisoldipine or nitrendipine. In other embodiments, othernon-limiting examples of cardiovascular drugs include atenolol,bucindolol, carvedilol, clonidine, doxazosin, indoramin, labetalol,methyldopa, metoprolol, nadolol, oxprenolol, phenoxybenzamine,phentolamine, pindolol, prazosin, propranolol, terazosin, timolol ortolazoline. In other embodiments, the cardiovascular drug may be, forexample, a statin, such as atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin orsimvastatin.

Methods of treating or preventing a neurodegenerative disease in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure are alsocontemplated. In some embodiments, the neurodegenerative disease maybeselected, for example, from the group consisting of Parkinson's disease,Alzheimer's disease, multiple sclerosis (MS), Huntington's disease andamyotrophic lateral sclerosis. In particular embodiments, theneurodegenerative disease is Alzheimer's disease. In particularembodiments, the neurodegenerative disease is MS, such as primaryprogressive, relapsing-remitting secondary progressive or progressiverelapsing MS. In some embodiments, the subject may be, for example, aprimate. In some embodiments, the subject may be a human.

In particular embodiments of methods of treating or preventing aneurodegenerative disease in a subject, comprising administering to thesubject a pharmaceutically effective amount of a compound of the presentdisclosure, the treatment suppresses the demyelination of neurons in thesubject's brain or spinal cord. In certain embodiments, the treatmentsuppresses inflammatory demyelination. In certain embodiments, thetreatment suppresses the transection of neuron axons in the subject'sbrain or spinal cord. In certain embodiments, the treatment suppressesthe transection of neurites in the subject's brain or spinal cord. Incertain embodiments, the treatment suppresses neuronal apoptosis in thesubject's brain or spinal cord. In certain embodiments, the treatmentstimulates the remyelination of neuron axons in the subject's brain orspinal cord. In certain embodiments, the treatment restores lostfunction after an MS attack. In certain embodiments, the treatmentprevents a new MS attack. In certain embodiments, the treatment preventsa disability resulting from an MS attack.

One general aspect of the present disclosure contemplates a method oftreating or preventing a disorder characterized by overexpression ofiNOS genes in a subject, comprising administering to the subject apharmaceutically effective amount of RTA 408, polymorphic forms, or apharmaceutical composition of the present disclosure.

Another general aspect of the present disclosure contemplates a methodof inhibiting IFN-γ-induced nitric oxide production in cells of asubject, comprising administering to said subject a pharmaceuticallyeffective amount of RTA 408, polymorphic forms, or a pharmaceuticalcomposition of the present disclosure.

Yet another general method of the present disclosure contemplates amethod of treating or preventing a disorder characterized byoverexpression of COX-2 genes in a subject, comprising administering tothe subject a pharmaceutically effective amount of RTA 408, polymorphicforms, or a pharmaceutical composition of the present disclosure.

Methods of treating renal/kidney disease (RKD) in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure are also contemplated. See U.S. Pat.No. 8,129,429, which is incorporated by reference herein. The RKD mayresult from, for example, a toxic insult. The toxic insult may resultfrom but not limited to, for example, an imaging agent or a drug. Thedrug may be a chemotherapeutic, for example. The RKD may result fromischemia/reperfusion injury, in certain embodiments. In certainembodiments, the RKD results from diabetes or hypertension. In someembodiments, the RKD may result from an autoimmune disease. The RKD maybe further defined as chronic RKD or acute RKD.

In certain methods of treating renal/kidney disease (RKD) in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure, the subject hasundergone or is undergoing dialysis. In certain embodiments, the subjecthas undergone or is a candidate to undergo kidney transplant. Thesubject may be a primate. The primate may be a human. The subject inthis or any other method may be, for example, a cow, horse, dog, cat,pig, mouse, rat or guinea pig.

Also contemplated by the present disclosure is a method for improvingglomerular filtration rate or creatinine clearance in a subject,comprising administering to the subject a pharmaceutically effectiveamount of RTA 408, polymorphic forms, or a pharmaceutical composition ofthe present disclosure.

In some embodiments, the pharmaceutical composition is administered in asingle dose per day. In other embodiments, the pharmaceuticalcomposition is administered in more than one dose per day. In someembodiments, the pharmaceutical composition is administered in apharmaceutically effective amount.

In some embodiments, the dose is from about 1 mg/kg to about 2000 mg/kg.In other embodiments, the dose is from about 3 mg/kg to about 100 mg/kg.In other embodiments, the dose is about 3, 10, 30, or 100 mg/kg.

In other embodiments, the pharmaceutical composition is administeredtopically. In some embodiments, the topical administration isadministered to the skin. In other embodiments, the topicaladministration is administered to the eye.

In other embodiments, the pharmaceutical composition is administeredorally. In other embodiments, the pharmaceutical composition isadministered intraocularly.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula does not mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. One or more German words can be found in the drawings,including “Masseänderung” and “temperatur”, which mean “change in mass”and “temperature”, respectively. The invention may be better understoodby reference to one of these drawings in combination with the detaileddescription of specific embodiments presented herein.

FIG. 1—Effect of RTA 408 on IFNγ-induced nitric oxide production andcell viability in RAW264.7 cells.

FIGS. 2 a & b—Effect of RTA 408 on antioxidant response element (ARE)activation: (a) NQO1-ARE luciferase activity; (b) GSTA2-ARE luciferaseactivity.

FIGS. 3 a-f—Relative Nrf2 GST ARE fold increase after cellular treatmentwith (a) RTA 402; (b) 63415 (RTA 408); (c) 63170; (d) 63171; (e) 63179;and (f) 63189. The graph also show the viability of the cells as assayedusing the WST1 cell proliferation reagent and measuring the absorbanceafter 1 hour. All drugs were administered in DMSO and cells were grownat 10,000 cells/well in 384-well plates in DMEM low glucose supplementedwith 10% FBS, 1% Penicillin Streptomycin, and 0.8 mg/mL Geneticin.

FIGS. 4 a-d—Effect of RTA 408 on Nrf2 target gene expression in HFL1lung fibroblasts. (a) NQO1; (b) HMOX1; (c) GCLM; (d) TXNRD1.

FIGS. 5 a-d—Effect of RTA 408 on Nrf2 target gene expression in BEAS-2Bbronchial epithelial cells. (a) NQO1; (b) HMOX1; (c) GCLM; (d) TXNRD1.

FIGS. 6 a & b—Effect of RTA 408 on Nrf2 target protein levels. (a)SH-SY5Y cells; (b) BV2 cells.

FIG. 7—Effect of RTA 408 on NQO1 enzymatic activity in RAW264.7 cells.

FIG. 8—Effect of RTA 408 on total glutathione levels in the AML-12hepatocyte cell line.

FIG. 9—Effect of RTA 408 on WST-1 absorbance as a marker of NADPH.

FIGS. 10 a-d—Effect of RTA 408 on expression of genes involved in NADPHsynthesis. (a) H6PD; (b) PGD; (c) TKT; (d) ME1.

FIGS. 11 a & b—(a) Effect of RTA 408 on TNF-α-induced activation of anNF-κB luciferase reporter in the mouse NIH3T3 cell line with WST1viability and WST1/2 viability overlaid. (b) TNF-α-induced activation ofan NF-κB luciferase reporter in the mouse NIH3T3 cell line. The graphshows relative fold change as a function of log change in RTA 408concentration.

FIG. 12—Effect of RTA 408 on TNF-α-induced activation of a NF-κBluciferase reporter construct.

FIGS. 13 a & b—(a) Effect of RTA 408 on TNF-α-induced activation of anNF-κB luciferase reporter in the human A549 cell line with WST1viability and WST1/2 viability overlaid. (b) TNF-α-induced activation ofan NF-κB luciferase reporter in the human A549 cell line. The graphshows relative fold change as a function of log change in RTA 408concentration.

FIG. 14—Effect of RTA 408 on TNF-α-induced phosphorylation of IκBα.

FIGS. 15 a-d—Effect of RTA 408 on transaminase gene expression: (a) ALT1(GPT1); (b) ALT2 (GPT2); (c) AST1 (GOT1); (d) AST1 (GOT2). Asterisksindicate a statistically-significant difference from the control group(*P<0.05; **P<0.01).

FIG. 16—Effect of RTA 408 on pyruvate levels in cultured muscle cells(*P<0.05).

FIG. 17—Effect of 63415 in a model of pulmonary LPS-mediatedinflammation (% change in pro-inflammatory cytokines relative to LPStreatment). Compound 63415 was administered QD×3 at Time 0, 24, and 48 hfollowed by LPS one h after the last dose of 63415 in female BALB/cmice. Animals were sacrificed 20 h after LPS administration. BALF wasexamined for pro-inflammatory cytokine expression. Compound 63415reduced pro-inflammatory cytokines in a dose-dependent manner, with peakreductions ranging from 50%-80% in TNF-α, IL-6, and IL-12.

FIGS. 18 a & b—Effect of RTA 408 on LPS-induced pulmonary inflammationin mice. (a) inflammatory cytokines; (b) Nrf2 targets. RTA 408 wasadministered to female BALB/c mice (n=10) QD×6 at Time 0, 24, 48, 72,96, and 120 h followed by LPS at 121 h with animals sacrificed at 141 h.Pro-inflammatory cytokine protein expression assayed in BALF. Nrf2biomarkers assayed in lung. Asterisks indicate a statisticallysignificant difference from the saline control group (*P<0.05; **P<0.01;***P<0.001).

FIGS. 19 a & b—Effect of 63415 on BALF infiltrates in bleomycin-inducedpulmonary inflammation: (a) BAL fluid cell count; (b) body weight.Compound 63415 was administered QD×39 on Days −10 to 28 in C57BL/6 mice.Bleomycin was given on Day 0. Daily weights were measured. BALF cellcounts were obtained at sacrifice. A notable reduction in inflammatoryinfiltrate was observed. No significant improvements in chronicinflammation score, interstitial fibrosis, or number of fibrotic fociwere observed.

FIGS. 20 a & b—Effect of RTA 408 on bleomycin-induced pulmonary fibrosisin rats: (a) PMN; (b) Hydroxyproline. Asterisks indicate a statisticallysignificant difference from the bleomycin control group (*P<0.05).

FIG. 21—Effect of RTA 408 on Nrf2 target enzymes in lungs from rats withbleomycin-induced pulmonary fibrosis. Asterisks indicate a statisticallysignificant difference from the saline control group (*P<0.05; **P<0.01;***P<0.001).

FIGS. 22 a-e—Effect of RTA 408 on cigarette smoke-induced COPD in mice.(a) KC; (b) IL-6; (c) TNF-α; (d) IFN-γ; (e) RANTES. RTA 408 (63415) wastested at dose levels of 3 mg/kg (low), 10 mg/kg (mid), and 30 mg/kg(high). An AIM analog (63355) was tested in the same study forcomparison. Asterisks indicate a statistically significant differenceform the CS control group.

FIG. 23—Effect of RTA 408 on Nrf2 target enzymes in lungs from mice withcigarette smoke-induced COPD. Asterisks indicate a statisticallysignificant difference from the saline control group (*P<0.05; **P<0.01;***P<0.001). Daggers represent a statistically significant differencefrom mice expose to cigarette smoke and administered vehicle (†P<0.05).

FIGS. 24 a-d—Effects of 63415 (RTA 408) on body weight in a BALB/c mousemodel of sepsis. LPS was administered to all animals on Day 0. (a) BodyWeight: 63415 (RTA 408); (b) Body Weight: RTA 405; (c) Systemic LPS: %Survival: 63415 (RTA 408); (d) Systemic LPS: % Survival: RTA 405. BothRTA 405 and 63415 (RTA 408) were administered QD×5 on Days −2 to 2.63415 (RTA 408) improved survival. Body weight as a function of time in63415-treated BALB/c mice serves as a model for sepsis.

FIG. 25—Effect of 63415 in a model of radiation-induced oral mucositis.RTA 405 or 63415 (RTA 408) was administered BID×20 on Days −5 to −1 andDays 1 to 15 to male Syrian Golden Hamsters. Radiation occurred on Day0. Mucositis scores range from 0 to 5 based on clinical manifestations(0: completely healthy; 1-2: light to severe erythema; 3-5: varyingdegrees of ulceration). 63415 improved mucositis at 30 mg/kg and 100mg/kg with up to a 36% reduction in ulceration.

FIG. 26—Effect of 63415 on Nrf2 target gene induction in a 14-day mousetoxicity study in C57BL/6 mice. mRNAs of Nrf2 target genes were assessedin livers of mice treated PO QD×14. Substantial increases in mRNAexpression for multiple Nrf2 target genes were observed and wereconsistent with tissue exposure.

FIGS. 27 a & b—Effect of 63415 on Nrf2 target gene induction in ratlivers: (a) Target genes; (b) Negative regulators. mRNAs of Nrf2 targetgenes were assessed in livers of rats treated PO QD×14.

FIGS. 28 a & b—Effect of 63415 on Nrf2 target genes in monkey tissues:(a) Liver; (b) Lung mRNAs of Nrf2 target genes were assessed in monkeystreated PO QD×14 using Panomics QuantiGene® 2.0 Plex technology.

FIGS. 29 a & b—Effect of 63415 on Nrf2 target enzyme activity in mouseliver: (a) NQO1 activity; (b) GST activity. Nrf2 target enzyme activitywas assessed in livers of mice treated PO QD×14. NQO1 and GST enzymeactivities were induced in a dose-dependent manner.

FIGS. 30 a & b—Effect of 63415 on Nrf2 target enzyme activity in ratliver: (a) NQO1 activity; (b) GST activity. Nrf2 target enzyme activitywas assessed in livers of rats treated PO QD×14. NQO1 and GST enzymeactivities were induced in a dose dependent manner.

FIGS. 31 a & b—Effects of 63415 on Nrf2 target enzyme activity inductionin various tissues of cynomolgus monkeys: (a) NQO1 activity; (b) GSRactivity.

FIGS. 32 a & b—RTA 408 concentration in mouse liver, lung, and brain,and NQO1 activity in mouse liver after 14 days of daily oraladministration. (a) Tissue distribution of RTA 408 in mice after 14 daysof daily oral administration. Data represent the mean±SD of RTA 408concentrations in tissue collected 4 h after the final dose of thestudy. Numbers above the error bars are representative of the mean. (b)Correlation of mouse liver RTA 408 content with NQO1 enzyme activity.Individual mouse liver RTA 408 liver content was plotted againstindividual enzyme activity from this report.

FIGS. 33 a & b—RTA 408 concentration in rat plasma, liver, lung, andbrain, and NQO1 activity in rat liver after 14 days of daily oraladministration. (a) Tissue distribution of RTA 408 in rats after 14 daysof daily oral administration. Data represent the mean±SD of RTA 408concentrations in tissue collected 4 h after the final dose of thestudy. Numbers above the error bars are representative of the mean. *Twovalues were excluded from the mean calculation due to being outliers,defined as values causing the set of data to fail the Shapiro-Wilknormality test. (b) Correlation of rat liver RTA 408 content with NQO1enzyme activity. Individual rat liver RTA 408 content was plottedagainst individual enzyme activity from this report. The tissues fromthe 100 mg/kg RTA 408 dose group were collected on Day 6, and theobserved toxicities in this group precluded liver NQO1 enzyme activityevaluations.

FIGS. 34 a & b—Effect of 63415 treatment on Nrf2 activation in monkeyPBMC: (a) PBMC NQO1 vs. Plasma Concentration; (b) Lung NQO1 vs. PBMCNQO1.

FIG. 35—Summary of 63415 14-day monkey toxicity study. All doses werewell-tolerated without adverse clinical signs. Clinical chemistry datasuggested no obvious toxicity.

FIG. 36—Effect of dosing time on plasma concentration of RTA 408 aftertopical ocular and oral administration. The plasma concentration of RTA408 was also measured after 5 days of daily topical ocularadministration of RTA 408 and determined to remain relatively consistentfrom the measurements taken after the first day.

FIGS. 37 a & b—Correlation of exposure to RTA 408 in monkey plasma withNQO1 and SRXN1 mRNA expression in PBMCs: (a) NQO1; (b) SRXN1.

FIG. 38—Concentration of RTA 408 in various tissues or fluids within theeye as a function of time after 5 days of topical ocular dosing. RTA 408concentration in plasma was also measured after topical ocularadministration.

FIG. 39—Effect of RTA 408 on the incidence of grade 3 dermatitis causedby acute radiation exposure for different concentrations of topicallyadministered RTA 408.

FIG. 40—Effect of RTA 408 on the incidence of grade 2 dermatitis causedby acute radiation exposure over a 30 day treatment course for differentconcentrations of topically administered RTA 408.

FIG. 41—Effect of RTA 408 on the incidence of grade 2 dermatitis causedby acute radiation exposure over a 28 day treatment course for differentconcentrations of orally administered RTA 408.

FIGS. 42 a & b—(a) Area under the curve analysis of clinical score ofthe dermatitis as a function of time for each of the different controlgroups including all of the animals used in the test. (b) Area under thecurve analysis of the clinical score of the dermatitis as a function ofthe duration of that score for each of the different control groupsincluding only animals that completed the entire 30 days in the trial.

FIG. 43—Average 1^(st) blind score of acute radiation dermatitis as afunction of time for untreated, untreated with no radiation exposure,vehicle only and three oral amounts of RTA 408 at 3, 10, and 30 mg/kg.The dermatitis score was based upon a scale that 0 was completelyhealthy, 1-2 exhibited mild to moderate erythema with minimal to slightdesquamation, 3-4 exhibited moderate to severe erythema anddesquamation, and 5 exhibited a frank ulcer.

FIG. 44—Mean score of the acute radiation dermatitis as a function oftime for untreated, untreated with no radiation exposure, vehicle only,and three oral amounts of RTA 408 at 3, 10, and 30 mg/kg measured everyother day from day 4 to day 30. The dermatitis score was based upon ascale that 0 was completely healthy, 1-2 exhibited mild to moderateerythema with minimal to slight desquamation, 3-4 exhibited moderate tosevere erythema and desquamation, and 5 exhibited a frank ulcer.

FIG. 45—Mean score of the acute radiation dermatitis as a function oftime for untreated, untreated with no radiation exposure, vehicle only,and three topical amounts of RTA 408 at 0.01%, 0.1%, and 1% measuredevery other day from day 4 to day 30. The dermatitis score was basedupon a scale that 0 was completely healthy, 1-2 exhibited mild tomoderate erythema with minimal to slight desquamation, 3-4 exhibitedmoderate to severe erythema and desquamation, and 5 exhibited a frankulcer.

FIG. 46—Clinical scores of fractional radiation dermatitis plottedversus time and changes in dermatitis score for each testing group. Thedermatitis score was based upon a scale that 0 was completely healthy,1-2 exhibited mild to moderate erythema with minimal to slightdesquamation, 3-4 exhibited moderate to severe erythema anddesquamation, and 5 exhibited a frank ulcer.

FIG. 47—Graph of the AUC analysis showing the dermatitis score(severity×days) for each of the testing groups over the entireobservation period. The dermatitis scores were assessed every two daysfrom day 4 to day 30 of the study.

FIGS. 48 a & b—(a) Graph of the absorbance at 595 nm for treatedprostate cancer cell line LNCaP showing relative cytotoxic effect oncells treated with a chemotherapeutic agent and RTA 408 versus RTA 408alone. (b) Graph of the absorbance at 595 nm for treated prostate cancercell line DU-145 showing relative cytotoxic effect on cells treated witha chemotherapeutic agent and RTA 408 versus RTA 408 alone.

FIG. 49—Black and white versions of color photographs of imaged miceshowing the luciferase activity of tumors for three mice: a controlanimal with no treatment, an animal subjected only to ionizing radiation(single dose, 18 Gy), and an animal given both ionizing radiation(single dose, 18 Gy, day 0) and RTA 408 (17.5 mg/kg i.p., once daily ondays −3 to −1, then single doses on days 1, 3, and 5). The colorsindicated by the arrows are indicative of intensity with the intensitiesbeing represented by red, yellow, green, and blue in order from highestto lowest intensity.

FIG. 50—Reduction of aqueous humor protein concentrations for differentformulations of RTA 408 (dark bars) compared to literature values forMaxiDex® (0.1% dexamethasone) and mapracorat (light bars) afterinduction of paracentesis.

FIG. 51—Dose-dependent suppression of NO in vivo by 63415. CD-1 mice(n=6) were dosed with DMSO or AIM by oral gavage. LPS (5 mg/kg) wasadministered 24 h later. Twenty-four hours after LPS administration,whole blood was collected for NO assay. NO inhibition was determined byGriess Reaction from reduced, de-proteinated plasma.

FIG. 52—Extensive distribution of 63415 (RTA 408) into mouse tissues.Mice were dosed PO QD×3 with either 25 mg/kg 63415 (RTA 408) or 25 mg/kgRTA 405. Blood (plasma and whole blood) and tissues (brain, liver, lung,and kidney) were collected 6 h after the last dose. Semi-quantitativeanalysis of drug content was performed. Notable levels were observed inthe CNS.

FIG. 53—NQO1 activity induction in mouse liver, lung, and kidney by63415. Mice were dosed PO QD×3 with 25 mg/kg. Tissues were collected 6 hafter the last dose, and analysis of NQO1 activity was performed.Meaningful activation of NQO1 was observed in multiple tissues.

FIG. 54—Summary of 63415 14-day mouse toxicity study. C57BL/6 mice weredosed PO QD×14. Endpoints included survival, weight, and clinicalchemistries. All animals survived to day 14. No significant weightchanges occurred compared to the vehicle group, and there was noevidence of toxicity at any dose based on clinical chemistries.

FIG. 55—Tissue distribution of 63415 from 14-day mouse toxicity study inC57BL/6 mice. Brain, lung, and liver samples were collected 4 h afterfinal dose and quantified for 63415 content using sensitive LC/MS/MSmethod. Exposures at 10 and 100 mg/kg in lung exceeded the in vitro IC₅₀for NO induction by 55- and 1138-fold, respectively. Exposure at 10 and100 mg/kg in brain exceeded the in vitro IC₅₀ for NO induction by 29-and 541-fold, respectively.

FIG. 56—Tissue distribution of 63415 in Sprague Dawley rats. Tissueswere collected four hours after final dosing on Day 14 or Day 6 (100mg/kg), extracted, and quantified for 63415 content using a sensitiveLC/MS/MS method. Compound 63415 distributes well into target tissues.Exposures at 10 mg/kg in lung and brain exceed the in vitro IC₅₀ for NOinhibition by 294- and 240-fold, respectively.

FIG. 57—Target tissue distribution of compound 63415 in cynomolgusmonkeys. Tissues were collected four hours after final dosing on Day 14.Compound 63415 content was extracted and quantified using a sensitiveLC/MS/MS method.

FIG. 58—FT-Raman spectrum (3400-50 cm⁻¹) of the sample PP415-P1, whichcorresponds to the amorphous form (Class 1).

FIG. 59—PXRD (1.5-55.5°2θ) pattern of the sample PP415-P1, whichcorresponds to the amorphous form (Class 1).

FIG. 60—TG-FTIR thermogram (25-350° C.) of the sample PP415-P1, whichcorresponds to the amorphous form (Class 1).

FIG. 61—¹H-NMR spectrum in DMSO-d₆ of the sample PP415-P1, whichcorresponds to the amorphous form (Class 1).

FIG. 62—DSC thermogram of the sample PP415-P1, which corresponds to theamorphous form (Class 1).

FIG. 63—DVS isotherm of the sample PP415-P1, which corresponds to theamorphous form (Class 1).

FIG. 64—FT-Raman spectrum of the sample PP415-P1, which corresponds tothe amorphous form (Class 1), after DVS measurement (top) is unchangedcompared to the material before the DVS measurement (bottom). Thespectra have been scaled and offset in the y-direction for the purposeof comparison.

FIG. 65—PXRD pattern of the sample PP415-P1, which corresponds to theamorphous form (Class 1), after DVS measurement (top) is unchangedcompared to the material before the DVS measurement (bottom). Thepatterns have not been scaled but are offset in the y-direction for thepurpose of comparison.

FIG. 66—PXRD pattern of the sample PP415-P40 (top) corresponds to thepattern of the solvate form (Class 2) (bottom, sample PP415-P19). Thepatterns have been scaled and offset in the y-direction for the purposeof comparison.

FIG. 67—PXRD patterns of the stability samples PP415-P2a (top),PP415-P3a (2^(nd) from top), PP415-P4a (middle), and PP415-P5a (2^(nd)from bottom), which corresponds to the amorphous form (Class 1), afterone week show no differences compared to the starting material at timepoint t₀ (bottom, sample PP415-P1). The patterns are not scaled but areoffset in the y-direction for the purpose of comparison.

FIG. 68—PXRD patterns of the stability samples PP415-P2b (top),PP415-P3b (2^(nd) from stop), PP415-P4b (middle), and PP415-P5b (2^(nd)from bottom), which corresponds to the amorphous form (Class 1), aftertwo weeks show no differences compared to the starting material at timepoint t₀ (bottom, sample PP415-P1). The patterns are not scaled but areoffset in the y-direction for the purpose of comparison.

FIG. 69—PXRD patterns of the stability samples PP415-P2c (top),PP415-P3c (2^(nd) from top), PP415-P4c (middle), and PP415-P5c (2^(nd)from bottom), which corresponds to the amorphous form (Class 1), afterfour weeks show no differences compared to the starting material at timepoint t0 (bottom, sample PP415-P1). The patterns are not scaled but areoffset in the y-direction for the purpose of comparison.

FIG. 70—FT-Raman spectra (2400-50 cm⁻¹) of samples of the solvate form(Class 2) (PP415-P7: top; PP415-P8: 2^(nd) from top; PP415-P9: 3^(rd)from top; PP415-P10: 4^(th) from top; PP415-P11: middle; PP415-P15:4^(th) from bottom; PP415-P17: 3^(rd) from bottom; PP415-P21: 2^(nd)from bottom; PP415-P24: bottom). The spectra have been scaled and offsetin the y-direction for the purpose of comparison.

FIG. 71—FT-Raman spectrum (1750-1000 cm⁻¹) of the solvate form (Class 2)(PP415-P7: top) clearly differs from the spectrum of the amorphous form(Class 1) (PP415-P1: bottom). The spectra have been scaled and offset inthe y-direction for the purpose of comparison.

FIG. 72—FT-Raman spectra (1750-1000 cm⁻¹) of class 2 (sample PP415-P19:top), class 3 (sample PP415-P6: 2^(nd) from top), class 4 (samplePP415-P13: 2^(nd) from bottom), and class 5 (sample PP415-P14: bottom)differ significantly from each other. The spectra have been scaled andoffset in the y-direction for the purpose of comparison.

FIG. 73—PXRD patterns (2-32°2θ) of samples of the solvate form (Class 2)(PP415-P7: top; PP415-P8: 2^(nd) from top; PP415-P10: 3^(rd) from top;PP415-P15: 4^(th) from top; PP415-P17: middle; PP415-P18: 4^(th) frombottom; PP415-P19: 3^(rd) from bottom; PP415-P21: 2^(nd) from bottom;PP415-P24: bottom). The patterns have been scaled and offset in they-direction for the purpose of comparison.

FIG. 74—PXRD patterns (11-21°2θ) of some samples of the solvate form(Class 2) (PP415-P7: top; PP415-P8: 2^(nd) from top; PP415-P10: middle;PP415-P21: 2^(nd) from bottom; PP415-P24: bottom). The patterns havebeen scaled and offset in the y-direction for the purpose of comparison.

FIG. 75—PXRD patterns (2-32°2θ) of class 2 (sample PP415-P19: top),class 3 (sample PP415-P6: 2^(nd) from top), class 4 (sample PP415-P13:2^(nd) from bottom), and class 5 (sample PP415-P14: bottom) aredistinctly different. The patterns have been scaled and offset in they-direction for the purpose of comparison.

FIG. 76—TG-FTIR thermogram of the sample PP415-P7, which corresponds toa solvate form (Class 2).

FIG. 77—TG-FTIR thermogram of the sample PP415-P21, which corresponds toa solvate form (Class 2).

FIG. 78—TG-FTIR thermogram of the sample PP415-P24, which corresponds toa solvate form (Class 2).

FIG. 79—TG-FTIR thermogram of the sample PP415-P29, which corresponds toa solvate form (Class 2).

FIG. 80—TG-FTIR thermogram of the sample PP415-P47, which corresponds toa solvate form (Class 2).

FIG. 81—TG-FTIR thermogram of the sample PP415-P48, which corresponds toa solvate form (Class 2).

FIG. 82—FT-Raman spectra (1800-700 cm⁻¹) of the solvate form (Class 2)(bottom, sample PP415-P7) and of the dried solvate form (Class 2) (top,sample PP415-P30) are similar and show only small differences which canhardly be distinguished within the graph. The spectra are scaled for thepurpose of comparison.

FIG. 83—PXRD pattern of the dried solvate form (Class 2), samplePP415-P30 (top) in comparison to the pattern of the solvate form (Class2), sample PP415-P7 (bottom). The patterns are not scaled but are offsetin the y-direction for the purpose of comparison.

FIG. 84—TG-FTIR thermogram of the dried sample PP415-P30, whichcorresponds to a solvate form (Class 2).

FIG. 85—FT-Raman spectrum of the dried sample PP415-P18 (light grey) isidentical to the spectrum of the original sample PP415-P15 (dark grey)are similar and show only small differences which can hardly bedistinguished within the graph. The spectra have been scaled for thepurpose of comparison.

FIG. 86—PXRD pattern of the dried sample PP415-P18 (top) shows smalldifferences from the pattern of the original sample PP415-P15 (bottom),although both solvate forms (Class 2). The patterns have been scaled andoffset in the y-direction for the purpose of comparison.

FIG. 87—TG-FTIR thermogram of the sample PP415-P18, which corresponds toa solvate form (Class 2).

FIG. 88—FT-Raman spectrum of the sample PP415-P17 (top) is nearlyidentical to the spectra of the dried samples PP415-P19 (middle) andPP415-P32 (bottom) and show only small differences which can hardly bedistinguished within the graph. The spectra have been scaled and offsetin the y-direction for the purpose of comparison.

FIG. 89—PXRD pattern of the dried sample PP415-P19 (middle) is differentfrom the pattern of the original sample PP415-P17 (top) but stillcorresponds to class 2 form. The pattern of the further dried samplePP415-P32 (bottom) shows broader peaks with a lower S/N ratio. Thematerial is less crystalline, but still corresponds to class 2 form. Thepatterns have been scaled and offset in the y-direction for the purposeof comparison.

FIG. 90—TG-FTIR thermogram of the sample PP415-P19, which corresponds toa solvate form (Class 2).

FIG. 91—TG-FTIR thermogram of the sample PP415-P32A, which correspondsto a solvate form (Class 2).

FIG. 92—FT-Raman spectrum of the sample PP415-P21 (top) is identical tothe spectra of the dried samples PP415-P28 (middle) and PP415-P34(bottom). The spectra have been scaled and offset in the y-direction forthe purpose of comparison.

FIG. 93—PXRD patterns of the dried samples PP415-P28 (middle) andPP415-P34 (bottom) show broader peaks with a lower S/N ratio, indicatinga lower crystallinity of the samples compared to the pattern of theoriginal sample PP415-P21 (top). The patterns are somewhat different butstill correspond to class 2 form. They have been scaled and offset inthe y-direction for the purpose of comparison.

FIG. 94—TG-FTIR thermogram of the dried sample PP415-P28, whichcorresponds to a solvate form (Class 2).

FIG. 95—TG-FTIR thermogram of the dried sample PP415-P34, whichcorresponds to a solvate form (Class 2).

FIG. 96—FT-Raman spectra (2400-50 cm⁻¹) of the samples of the solvateform (Class 3) (PP415-P6: top; PP415-P12: middle; PP415-P20: bottom).The spectra have been scaled and offset in the y-direction for thepurpose of comparison.

FIG. 97—FT-Raman spectra (1750-1000 cm⁻¹) of the samples of the solvateform (Class 3) (PP415-P6: top; PP415-P12: 2^(nd) from top; PP415-P20:2^(nd) from bottom) are very similar to each other with only smalldifferences, e.g., at 1690 cm⁻¹, but are clearly different from class 1(PP415-P1: bottom). The spectra have been scaled and offset in they-direction for the purpose of comparison.

FIG. 98—PXRD patterns (2-32°2θ) of the samples of the solvate form(Class 3) (PP415-P6: top; PP415-P12: middle; PP415-P20: bottom). Thepatterns have been scaled and offset in the y-direction for the purposeof comparison.

FIG. 99—PXRD patterns (13.5-18.5°2θ) of the samples of solvate form(Class 3) (PP415-P6: top; PP415-P12: middle; PP415-P20: bottom) showsmall differences. The patterns have been scaled and offset in they-direction for the purpose of comparison.

FIG. 100—TG-FTIR thermogram of the sample PP415-P6, which corresponds tothe solvate form (Class 3).

FIG. 101—TG-FTIR thermogram of the sample PP415-P12, which correspondsto the solvate form (Class 3).

FIG. 102—TG-FTIR thermogram of the dried solvate form (Class 3), samplePP415-P25.

FIG. 103—TG-FTIR thermogram of the further dried solvate form (Class 3),sample PP415-P33.

FIG. 104—FT-Raman spectra (1800-700 cm⁻¹) of the solvate form (Class 3)(top, sample PP415-P6), of the dried solvate form (Class 3) (middle,sample PP415-P25), and of the further dried solvate form (Class 3)(bottom, sample PP415-P33) are identical. The spectra have been scaledand offset in the y-direction for the purpose of comparison.

FIG. 105—PXRD patterns (4-24°2θ) of the solvate form (Class 3) (top,sample PP415-P6), of the dried solvate form (Class 3) (middle, samplePP415-P25), and of the further dried solvate form (Class 3) (bottom,sample PP415-P33). The patterns have been scaled and offset in they-direction for the purpose of comparison.

FIG. 106—TG-FTIR thermogram of the sample PP415-P13, which correspondsto an acetonitrile solvate form (Class 4).

FIG. 107—FT-Raman spectra (1800-700 cm⁻¹) of the acetonitrile solvateform (Class 4) (dark grey, sample PP415-P13) and of the dried materialof an acetonitrile solvate form (Class 4) (light grey, sample PP415-P26)are identical and overlay perfectly. The spectra have been scaled forpurposes of comparison.

FIG. 108—PXRD pattern of the dried acetonitrile solvate form (Class 4),sample PP415-P26 (bottom), in comparison to the reference pattern of theacetonitrile solvate form (Class 4), sample PP415-P13 (top). Thepatterns have not been scaled but were offset in the y-direction forpurposes of comparison.

FIG. 109—TG-FTIR thermogram of the dried acetonitrile solvate form(Class 4), sample PP415-P26.

FIG. 110—FT-Raman spectra (1800-700 cm⁻¹) of the acetonitrile solvateform (Class 4) (top, sample PP415-P35), and of the dried acetonitrilesolvate form (Class 4) (middle, sample PP415-P36 and bottom, samplePP415-P37) correspond to each other. The spectra have been scaled andoffset in the y-direction for purposes of comparison.

FIG. 111—PXRD patterns (4-24°2θ) of the acetonitrile solvate form (Class4) (top, sample PP415-P35) and of the dried acetonitrile solvate form(Class 4) (middle, sample PP415-P36 and bottom, sample PP415-P37) agreewith each other. The patterns have been scaled and offset in they-direction for the purpose of comparison.

FIG. 112—TG-FTIR thermogram of the dried acetronitrile solvate form(Class 4), sample PP415-P36.

FIG. 113—TG-FTIR thermogram of the dried acetonitrile solvate form(Class 4), sample PP415-P37.

FIG. 114—DVS isotherm of the desolvated acetronitrile solvate form(Class 4), sample PP415-P37.

FIG. 115—PXRD pattern of the sample PP415-P37, an acetonitrile solvateform (Class 4) after the DVS measurement (bottom) is unchanged comparedto the material before the DVS measurement (top). The patterns have notbeen scaled but are offset in the y-direction for the purpose ofcomparison.

FIG. 116—DSC thermogram of the desolvated acetonitrile solvate form(Class 4) (sample PP415-P37).

FIG. 117—DSC thermogram of a ˜1:1 mixture of the amorphous form (Class1), sample PP415-P1, with the desolvated acetonitrile solvate form(Class 4), sample PP415-P36.

FIG. 118—DSC thermogram of a ˜1:1 mixture of the amorphous form (Class1), sample PP415-P1, with the desolvated acetonitrile solvate form(Class 4), sample PP415-P36 (experiment number: PP415-P39). The heatingscan (Step 1) was stopped for 30 min at 173° C. (Step 2) and thenresumed (Step 3).

FIG. 119—TG-FTIR thermogram of the sample PP415-P14, which correspondsto a THF solvate form (Class 5).

FIG. 120—FT-Raman spectra (1800-1100 cm⁻¹) of a THF solvate form (Class5) (dashed line, sample PP415-P14), dried material of a THF solvate form(Class 5) (dotted line, sample PP415-P27), and of the amorphous form(Class 1) (solid line, sample PP415-P1). The spectra have been scaledfor the purpose of comparison and show small changes in magnitude butlittle corresponding change in spectral shape.

FIG. 121—PXRD pattern of the dried THF solvate form (Class 5), samplePP415-P27 (top) in comparison to the pattern of the THF solvate form(Class 5), sample PP415-P14 (bottom). The patterns have not been scaledbut are offset in the y-direction for the purpose of comparison.

FIG. 122—TG-FTIR thermogram of the sample PP415-P27, which correspondsto a dried THF solvate (Class 5).

FIG. 123—PXRD pattern of sample PP415-P41 (top) corresponds to thepattern of the THF solvate form (Class 5) (middle, sample PP415-P14) andnot to the pattern of the heptane solvate form, (Class 2) (bottom,sample PP415-P19). The patterns have been scaled and offset in they-direction for purposes of comparison.

FIG. 124—PXRD pattern of sample PP415-P45 (top) corresponds to thepattern of the THF solvate form (Class 5) (middle, sample PP415-P14) andnot to the pattern of the heptane solvate form (Class 2) (bottom, samplePP415-P19). The patterns have been scaled and offset in the y-directionfor the purpose of comparison.

FIG. 125—PXRD pattern of sample PP415-P41 (top) corresponds to a THFsolvate form (Class 5). After drying sample PP415-P41 for 1 day (2^(nd)from top, sample: PP415-P44), the material is mainly amorphous. Somebroad peaks with low intensity remain. After further drying overnight(2^(nd) from bottom, sample PP415-P44a) the intensity of these broadpeaks is further reduced. The amorphous form (Class 1) is shown as areference (bottom, sample: PP415-P42). The patterns have not been scaledbut are offset in the y-direction for the purpose of comparison.

FIG. 126—TG-FTIR thermogram of the sample PP415-P44a, which correspondsto the amorphous form (Class 1).

FIG. 127—PXRD pattern of sample PP415-P45 (top) corresponds to a THFsolvate form (Class 5). After drying sample PP415-P45 for 1 day (2^(nd)from top, sample PP415-P46), the material is mainly amorphous. Somebroad peaks with low intensity remain. After a total of 4 days of drying(2^(nd) from bottom, sample PP415-P46a), the pattern remains unchanged.The amorphous form (Class 1) is shown as reference (bottom, samplePP415-P42). The patterns have not been scaled but are offset in they-direction for the purpose of comparison.

FIG. 128—TG-FTIR thermogram of the sample PP415-P46a, which correspondsto the amorphous form (Class 1).

FIG. 129—PXRD pattern of sample PP415-P42 (top) corresponds to thepattern of the amorphous form (Class 1) (bottom, sample PP415-P1). Thepatterns have been scaled and offset in the y-direction for the purposeof comparison.

FIG. 130—PXRD pattern of sample PP415-P43 (top) corresponds to thepattern of the isostructural solvate form (Class 2) (bottom, samplePP415-P19) and not to the pattern of the THF solvate form (Class 5)(middle, sample PP415-P14). The patterns have been scaled and offset inthe y-direction for the purpose of comparison.

FIG. 131—PXRD patterns of samples PP415-P47 (top) and PP415-P48 (middle)correspond essentially to the pattern of the isostructural solvate forms(Class 2) (bottom, sample PP415-P19), although there are somedifferences. The patterns have been scaled and offset in the y-directionfor the purpose of comparison.

FIG. 132—PXRD pattern of sample PP415-P49 (top) corresponds to thepattern of the amorphous form (Class 1) (bottom, sample PP415-P1). Thepatterns have been scaled and offset in the y-direction for the purposeof comparison.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides in one aspect the compound:

-   -   N-((4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b,9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octadecahydropicen-4a-yl)-2,2-difluoropropanamide,        which is also referred to herein as RTA 408, 63415, or PP415. In        other non-limiting aspects, the present invention also provides        polymorphic forms thereof, including solvates thereof. In other        non-limiting aspects, the invention also provides        pharmaceutically acceptable salts thereof. In other non-limiting        aspects, there are also provided methods for preparation,        pharmaceutical compositions, and kits and articles of        manufacture of these compounds and polymorphic forms thereof.

I. Definitions

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “carbonyl” means —C(═O)—; “carboxy”means —C(═O)OH (also written as —COOH or —CO₂H); “halo” meansindependently —F, —Cl, —Br or —I; “amino” means —NH₂; “hydroxyamino”means —NHOH; “nitro” means —NO₂; imino means ═NH; “cyano” means —CN;“isocyanate” means —N═C═O; “azido” means —N₃; in a monovalent context“phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; in adivalent context “phosphate” means —OP(O)(OH)O— or a deprotonated formthereof; “mercapto” means —SH; and “thio” means ═S; “sulfonyl” means—S(O)₂—; and “sulfinyl” means —S(O)—. Any undefined valency on an atomof a structure shown in this application implicitly represents ahydrogen atom bonded to the atom.

In the context of this disclosure, the formulas:

represent the same structures. When a dot is drawn on a carbon, the dotindicates that the hydrogen atom attached to that carbon is coming outof the plane of the page.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects. When used in the context of X-raypowder diffraction, the term “about” is used to indicate a value of±0.2°2θ from the reported value, preferably a value of ±0.1°2θ from thereported value. When used in the context of differential scanningcalorimetry or glass transition temperatures, the term “about” is usedto indicate a value of ±10° C. relative to the maximum of the peak,preferably a value of ±2° C. relative to the maximum of the peak. Whenused in other context, the term “about” is used to indicate a value of±10% of the reported value, preferably a value of ±5% of the reportedvalue. It is to be understood that, whenever the term “about” is used, aspecific reference to the exact numerical value indicated is alsoincluded.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment for the disease.

The term “halo peak” in the context of X-ray powder diffraction wouldmean a broad peak, often spanning >10°2θ in an X-ray powderdiffractogram, typically characteristic of an amorphous solid or system.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose that is 50%of the maximum response obtained. This quantitative measure indicateshow much of a particular drug or other substance (inhibitor) is neededto inhibit a given biological, biochemical, or chemical process (orcomponent of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a non-human animal. In certainembodiments, the patient or subject is a primate. In certainembodiments, the patient or subject is a human. Non-limiting examples ofhuman subjects are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- anddicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids,benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid,cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonicacid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid,glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid,lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid,mandelic acid, methanesulfonic acid, muconic acid,o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonicacid, phenyl-substituted alkanoic acids, propionic acid,p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid,succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylaceticacid, and the like. Pharmaceutically acceptable salts also include baseaddition salts which may be formed when acidic protons present arecapable of reacting with inorganic or organic bases. Acceptableinorganic bases include sodium hydroxide, sodium carbonate, potassiumhydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organicbases include ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine and the like. It should be recognizedthat the particular anion or cation forming a part of any salt of thisinvention is not critical, so long as the salt, as a whole, ispharmacologically acceptable. Additional examples of pharmaceuticallyacceptable salts and their methods of preparation and use are presentedin Handbook of Pharmaceutical Salts Properties, and Use (P. H. Stahl &C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present invention. The prodrug itselfmay or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylenebis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methane-sulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexyl-sulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2 n, where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diastereomers can be resolved or separated usingtechniques known in the art. It is contemplated that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≦15%, morepreferably ≦10%, even more preferably ≦5%, or most preferably ≦1% ofanother stereoisomer(s).

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

II. RTA 408 and Synthetic Methods

RTA 408 can be prepared according to the methods described in theExamples section below. These methods can be further modified andoptimized using the principles and techniques of organic chemistry asapplied by a person skilled in the art. Such principles and techniquesare taught, for example, in March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure (2007), which is incorporated byreference herein.

It should be recognized that the particular anion or cation forming apart of any salt of this invention is not critical, so long as the salt,as a whole, is pharmacologically acceptable. Additional examples ofpharmaceutically acceptable salts and their methods of preparation anduse are presented in Handbook of Pharmaceutical Salts: Properties, andUse (2002), which is incorporated herein by reference.

RTA 408 may also exist in prodrug form. Since prodrugs are known toenhance numerous desirable qualities of pharmaceuticals, e.g.,solubility, bioavailability, manufacturing, etc., the compounds employedin some methods of the invention may, if desired, be delivered inprodrug form. Thus, the invention contemplates prodrugs of compounds ofthe present invention as well as methods of delivering prodrugs.Prodrugs of the compounds employed in the invention may be prepared bymodifying functional groups present in the compound in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compound. Accordingly, prodrugs include, forexample, compounds described herein in which a hydroxy, amino, orcarboxy group is bonded to any group that, when the prodrug isadministered to a patient, cleaves to form a hydroxy, amino, orcarboxylic acid, respectively.

RTA 408 may contain one or more asymmetrically-substituted carbon ornitrogen atoms, and may be isolated in optically active or racemic form.Thus, all chiral, diastereomeric, racemic form, epimeric form, and allgeometric isomeric forms of a structure are intended, unless thespecific stereochemistry or isomeric form is specifically indicated. RTA408 may occur as racemates and racemic mixtures, single enantiomers,diastereomeric mixtures and individual diastereomers. In someembodiments, a single diastereomer is obtained. The chiral centers ofRTA 408 according to the present invention can have the S or the Rconfiguration.

In addition, atoms making up RTA 408 of the present invention areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C. Similarly, it is contemplated that one or morecarbon atom(s) of a compound of the present invention may be replaced bya silicon atom(s). Furthermore, it is contemplated that one or moreoxygen atom(s) of RTA 408 may be replaced by a sulfur or seleniumatom(s).

RTA 408 and polymorphic form thereof may also have the advantage thatthey may be more efficacious than, be less toxic than, be longer actingthan, be more potent than, produce fewer side effects than, be moreeasily absorbed than, and/or have a better pharmacokinetic profile(e.g., higher oral bioavailability and/or lower clearance) than, and/orhave other useful pharmacological, physical, or chemical advantagesover, compounds known in the prior art for use in the indications statedherein.

III. Polymorphic Forms of RTA 408

In some embodiments, the present invention provides different solidforms of RTA 408, including solvates thereof. A preformulation andpreliminary polymorphism study was performed, and RTA 408 was found tohave a high tendency for solvate formation. Crystalline forms of classes2, 3, 4, and 5 are consistent with solvates. For a description of theclasses, see Table 1 below. Attempts to dry classes 2 and 3 (two groupsof isostructural solvates) were not successful, which is consistent withtightly bound solvent molecules. In some embodiments, drying of a class4 solid (acetonitrile solvate) led to an isostructural desolvated form.In some embodiments, drying of a class 5 solid (THF solvate) resulted inthe amorphous form class 1. Non-solvated forms of RTA 408 include theamorphous form (class 1) and the crystalline desolvated solvate of class4 (isostructural to the class 4 acetonitrile solvate). In someembodiments, the amorphous form has a high glass transition withT_(g)≈153° C. (ΔCp=0.72 J/g° C.) and is only slightly hygroscopic(Δm=+0.4% 50%→85% r.h.). In some embodiments, the amorphous form isstable for at least four weeks under elevated temperature and humidityconditions (i.e., open at 40° C./˜75% r.h. or closed at 80° C.). In someembodiments, the amorphous form (class 1) was successfully prepared fromclass 2 material in a two-step process (transformation into class 5 andsubsequent drying of class 5 to obtain the amorphous form), as well asin a direct one-step method (precipitation from an acetone solution in acold water bath). The crystalline desolvated solvate of class 4(isostructural to the class 4 solvate) is slightly hygroscopic (massgain of ˜0.7 wt.-% from 50% r.h. to 85% r.h.) and has a possible meltingpoint at 196.1° C. (ΔH=29.31 J/g).

A sample of the amorphous form of 63415, class 1, was characterized byFT-Raman spectroscopy, PXRD, TG-FTIR, Karl Fischer titration, ¹H-NMR,DSC, and DVS (see Examples section for additional details). The samplewas found to contain ˜0.9 wt.-% EtOH with traces of H₂O (according tothe TG-FTIR). A water content of 0.5 wt.-% was determined by KarlFischer titration. DSC shows a high glass transition temperature withT_(g)≈153° C. (ΔC_(p)=0.72 J/g° C.). According to DVS, the material isslightly hygroscopic (Δm=+0.4% 50%→85% r.h.). No crystallization wasobserved in the DSC or DVS experiments.

The chemical stability of the amorphous form was investigated in organicsolvents, including acetone, EtOAc, MeOH, and MeCN, as well as differentaqueous media (e.g., 1% aq. Tween 80, 1% aq. SDS, 1% aq. CTAB) at aconcentration of 1 mg/mL at time points 6 h, 24 h, 2 d, and 7 d.Decomposition ≧1% was observed only for solutions in MeCN after 7 daysand for suspensions in the 1% aqueous Tween 80 medium (at all timespoints at 254 nm and after 24 h, 2 d, and 7 d at 242 nm).

In addition, the stability of the amorphous form was investigated bystorage under elevated temperature and humidity conditions (open at 25°C./62% r.h. and 40° C./75% r.h. and closed at 60° C. and 80° C.). Afterone week, two weeks, and four weeks, the stored samples were analyzed byPXRD. None of the samples differed from the amorphous starting material.

More than 30 crystallization and drying experiments were carried out,including suspension equilibration, slow cooling, evaporation, andprecipitation. Four new crystalline forms were obtained (classes 2, 3,4, and 5) in addition to the amorphous form (class 1).

The four new forms (classes 2, 3, 4, and 5) were characterized byFT-Raman spectroscopy, PXRD, and TG-FTIR. All forms correspond tosolvates (Table 1). Drying experiments under vacuum or N₂ flow werecarried out with the aim to obtain a crystalline, non-solvated form of63415.

TABLE 1 Summary of Obtained Classes Result of Class CharacteristicsDrying Experiments Class 1 amorphous form — Class 2 isostructuralsolvates (e.g., heptane) drying not successful Class 3 isostructuralsolvates (e.g., ethanol) drying not successful Class 4 MeCN solvate &desolvated solvate drying successful, structure unchanged Class 5 THFsolvate drying resulted in amorphous form

Class 2: Most crystallization experiments that were conducted resultedin solid material of class 2 (see Examples section below). Its membersmay correspond to isostructural, non-stoichiometric (<0.5 eq.) solvates(of heptane, cyclohexane, isopropyl ether, 1-butanol, triethylamine, andpossibly other solvents, such as hexane, other ethers, etc.) withtightly bound solvent molecules. The Raman spectra and PXRD patternswithin this class are very similar to each other, thus the structuresmight be essentially identical with only small differences due to thedifferent solvents that were incorporated.

Drying experiments on class 2 samples have not resulted in acrystalline, non-solvated form. Even elevated temperatures (80° C.) anda high vacuum (<1×10⁻³ mbar) could not remove the tightly bound solventmolecules completely; a solvent content of >2 wt.-% always remained. Thecrystallinity of these partially dried samples is reduced, but neithertransformation into a different structure nor substantial amorphizationwas observed.

Class 3: Solid material of class 3 may be obtained from severalcrystallizations (see Examples section below). The samples of class 3are likely isostructural solvates of 2PrOH, EtOH, and probably acetonewith tightly bound solvent molecules. They could correspond to eitherstoichiometric hemisolvates or non-stoichiometric solvates with asolvent content of ˜0.5 eq. As with class 2, the Raman spectra and PXRDpatterns within this class are very similar to each other, indicatingsimilar structures that incorporate different solvents.

Similar to class 2, drying experiments were not successful. The verytightly bound solvent molecules could only partially be removed (i.e.,˜5.4 wt.-% to ˜4.8 wt.-% after up to 3 d at 1×10⁻³ mbar and 80° C.). ThePXRD patterns remained unchanged.

Class 4 may be obtained from a 7:3 MeCN/H₂O solvent system (see Examplessection below). It most likely corresponds to a crystalline acetonitrilehemisolvate. By drying (under vacuum or N₂ flow at elevatedtemperatures) most of the solvent molecules could be removed withoutchanging or destroying the crystal structure (PXRD remained unchanged).Thus, a crystalline, non-solvated form (or rather desolvated solvate)was obtained. It is slightly hygroscopic (mass gain of ˜0.7 wt.-% from50% r.h. to 85% r.h.) and has a possible melting point at 196.1° C.(ΔH=29.31 J/g).

Class 5 may be obtained from an ˜1:1 THF/H₂O solvent system. Class 5contains bound THF (and maybe H₂O). As the content of the two componentscannot be readily quantified separately, the exact nature of thiscrystalline solvate has not been determined.

Drying of class 5 resulted in significant desolvation and transformationin the direction of the amorphous form (class 1). In some embodiments,the amorphous form of RTA 408 may be prepared by suspending class 2heptane solvate in 1:1 THF/H₂O to form a class 5 solid, followed bydrying and amorphization.

Experiments with the aim of preparing the amorphous form (class 1) werecarried out using class 2 starting material. Mainly amorphous material(class 1) was prepared starting from class 2 material in a two-stepprocess via class 5 on a 100-mg and 3-g scale (drying at 100 mbar, 80°C., several days). The preparation of fully amorphous material (class 1)was found to be possible in a one-step process avoiding the solvent THFby direct precipitation of the amorphous form (class 1) from an acetonesolution of class 2 material in a cold water bath.

IV. Diseases Associated with Inflammation and/or Oxidative Stress

Inflammation is a biological process that provides resistance toinfectious or parasitic organisms and the repair of damaged tissue.Inflammation is commonly characterized by localized vasodilation,redness, swelling, and pain, the recruitment of leukocytes to the siteof infection or injury, production of inflammatory cytokines, such asTNF-α and IL-1, and production of reactive oxygen or nitrogen species,such as hydrogen peroxide, superoxide, and peroxynitrite. In laterstages of inflammation, tissue remodeling, angiogenesis, and scarformation (fibrosis) may occur as part of the wound healing process.Under normal circumstances, the inflammatory response is regulated,temporary, and is resolved in an orchestrated fashion once the infectionor injury has been dealt with adequately. However, acute inflammationcan become excessive and life-threatening if regulatory mechanisms fail.Alternatively, inflammation can become chronic and cause cumulativetissue damage or systemic complications. Based at least on the evidencepresented herein, RTA 408 can be used in the treatment or prevention ofinflammation or diseases associated with inflammation.

Many serious and intractable human diseases involve dysregulation ofinflammatory processes, including diseases such as cancer,atherosclerosis, and diabetes, which were not traditionally viewed asinflammatory conditions. In the case of cancer, the inflammatoryprocesses are associated with processes that include tumor formation,progression, metastasis, and resistance to therapy. In some embodiments,RTA 408 may be used in the treatment or prevention of cancers includinga carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma,multiple myeloma, or seminoma, or cancer of the bladder, blood, bone,brain, breast, central nervous system, cervix, colon, endometrium,esophagus, gall bladder, genitalia, genitourinary tract, head, kidney,larynx, liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary,pancreas, prostate, skin, spleen, small intestine, large intestine,stomach, testicle, or thyroid. Atherosclerosis, long viewed as adisorder of lipid metabolism, is now understood to be primarily aninflammatory condition, with activated macrophages playing an importantrole in the formation and eventual rupture of atherosclerotic plaques.Activation of inflammatory signaling pathways has also been shown toplay a role in the development of insulin resistance, as well as in theperipheral tissue damage associated with diabetic hyperglycemia.Excessive production of reactive oxygen species and reactive nitrogenspecies, such as superoxide, hydrogen peroxide, nitric oxide, andperoxynitrite, is a hallmark of inflammatory conditions. Evidence ofdysregulated peroxynitrite production has been reported in a widevariety of diseases (Szabo et al., 2007; Schulz et al., 2008;Forstermann, 2006; Pall, 2007).

Autoimmune diseases such as rheumatoid arthritis, lupus, psoriasis, andmultiple sclerosis involve inappropriate and chronic activation ofinflammatory processes in affected tissues, arising from dysfunction ofself vs. non-self recognition and response mechanisms in the immunesystem. In neurodegenerative diseases such as Alzheimer's andParkinson's diseases, neural damage is correlated with activation ofmicroglia and elevated levels of pro-inflammatory proteins, such asinducible nitric oxide synthase (iNOS). Chronic organ failure, such asrenal failure, heart failure, liver failure, and chronic obstructivepulmonary disease, is closely associated with the presence of chronicoxidative stress and inflammation, leading to the development offibrosis and eventual loss of organ function. Oxidative stress invascular endothelial cells, which line major and minor blood vessels,can lead to endothelial dysfunction and is believed to be an importantcontributing factor in the development of systemic cardiovasculardisease, complications of diabetes, chronic kidney disease and otherforms of organ failure, and a number of other aging-related diseases,including degenerative diseases of the central nervous system and theretina.

Many other disorders involve oxidative stress and inflammation inaffected tissues, including inflammatory bowel disease; inflammatoryskin diseases; mucositis and dermatitis related to radiation therapy andchemotherapy; eye diseases, such as uveitis, glaucoma, maculardegeneration, and various forms of retinopathy; transplant failure andrejection; ischemia-reperfusion injury; chronic pain; degenerativeconditions of the bones and joints, including osteoarthritis andosteoporosis; asthma and cystic fibrosis; seizure disorders; andneuropsychiatric conditions, including schizophrenia, depression,bipolar disorder, post-traumatic stress disorder, attention deficitdisorders, autism-spectrum disorders, and eating disorders, such asanorexia nervosa. Dysregulation of inflammatory signaling pathways isbelieved to be a major factor in the pathology of muscle wastingdiseases, including muscular dystrophy and various forms of cachexia.

A variety of life-threatening acute disorders also involve dysregulatedinflammatory signaling, including acute organ failure involving thepancreas, kidneys, liver, or lungs, myocardial infarction or acutecoronary syndrome, stroke, septic shock, trauma, severe burns, andanaphylaxis.

Many complications of infectious diseases also involve dysregulation ofinflammatory responses. Although an inflammatory response can killinvading pathogens, an excessive inflammatory response can also be quitedestructive and in some cases can be a primary source of damage ininfected tissues. Furthermore, an excessive inflammatory response canalso lead to systemic complications due to overproduction ofinflammatory cytokines, such as TNF-α and IL-1. This is believed to be afactor in mortality arising from severe influenza, severe acuterespiratory syndrome, and sepsis.

The aberrant or excessive expression of either iNOS or cyclooxygenase-2(COX-2) has been implicated in the pathogenesis of many diseaseprocesses. For example, it is clear that NO is a potent mutagen (Tamirand Tannebaum, 1996), and that nitric oxide can also activate COX-2(Salvemini et al., 1994). Furthermore, there is a marked increase iniNOS in rat colon tumors induced by the carcinogen, azoxymethane(Takahashi et al., 1997). A series of synthetic triterpenoid analogs ofoleanolic acid have been shown to be powerful inhibitors of cellularinflammatory processes, such as the induction by IFN-γ of induciblenitric oxide synthase (iNOS) and of COX-2 in mouse macrophages. SeeHonda et al. (2000a), Honda et al. (2000b), and Honda et al. (2002),which are all incorporated herein by reference.

In one aspect, RTA 408 disclosed herein is in part characterized by itsability to inhibit the production of nitric oxide in macrophage-derivedRAW 264.7 cells induced by exposure to γ-interferon. RTA 408 is furthercharacterized by the ability to induce the expression of antioxidantproteins, such as NQO1, and reduce the expression of pro-inflammatoryproteins, such as COX-2 and inducible nitric oxide synthase (iNOS).These properties are relevant to the treatment of a wide array ofdiseases and disorders involving oxidative stress and dysregulation ofinflammatory processes, including cancer, complications from localizedor total-body exposure to ionizing radiation, mucositis and dermatitisresulting from radiation therapy or chemotherapy, autoimmune diseases,cardiovascular diseases, including atherosclerosis, ischemia-reperfusioninjury, acute and chronic organ failure, including renal failure andheart failure, respiratory diseases, diabetes and complications ofdiabetes, severe allergies, transplant rejection, graft-versus-hostdisease, neurodegenerative diseases, diseases of the eye and retina,acute and chronic pain, degenerative bone diseases, includingosteoarthritis and osteoporosis, inflammatory bowel diseases, dermatitisand other skin diseases, sepsis, burns, seizure disorders, andneuropsychiatric disorders.

In another aspect, RTA 408 may be used for treating a subject having acondition such as eye diseases. For example, uveitis, maculardegeneration (both the dry form and wet form), glaucoma, diabeticmacular edema, blepharitis, diabetic retinopathy, diseases and disordersof the corneal endothelium such as Fuchs endothelial corneal dystrophy,post-surgical inflammation, dry eye, allergic conjunctivitis and otherforms of conjunctivitis are non-limiting examples of eye diseases thatcould be treated with RTA 408.

In another aspect, RTA 408 may be used for treating a subject having acondition such as skin diseases or disorders. For example, dermatitis,including allergic dermatitis, atopic dermatitis, dermatitis due tochemical exposure, and radiation-induced dermatitis; thermal or chemicalburns; chronic wounds including diabetic ulcers, pressure sores, andvenous ulcers; acne; alopecia including baldness and drug-inducedalopecia; other disorders of the hair follicle; epidermolysis bullosa;sunburn and its complications; disorders of skin pigmentation includingvitiligo; aging-related skin conditions; post-surgical wound healing;prevention or reduction of scarring from skin injury, surgery, or burns;psoriasis; dermatological manifestations of autoimmune diseases orgraft-versus host disease; prevention or treatment of skin cancer;disorders involving hyperproliferation of skin cells such ashyperkeratosis is a non-limiting example of skin diseases that could betreated with RTA 408.

Without being bound by theory, the activation of theantioxidant/anti-inflammatory Keap1/Nrf2/ARE pathway is believed to beimplicated in both the anti-inflammatory and anti-carcinogenicproperties of the compound disclosed herein.

In another aspect, RTA 408 may be used for treating a subject having acondition caused by elevated levels of oxidative stress in one or moretissues. Oxidative stress results from abnormally high or prolongedlevels of reactive oxygen species, such as superoxide, hydrogenperoxide, nitric oxide, and peroxynitrite (formed by the reaction ofnitric oxide and superoxide). The oxidative stress may be accompanied byeither acute or chronic inflammation. The oxidative stress may be causedby mitochondrial dysfunction, by activation of immune cells, such asmacrophages and neutrophils, by acute exposure to an external agent,such as ionizing radiation or a cytotoxic chemotherapeutic agent (e.g.,doxorubicin), by trauma or other acute tissue injury, byischemia/reperfusion, by poor circulation or anemia, by localized orsystemic hypoxia or hyperoxia, by elevated levels of inflammatorycytokines and other inflammation-related proteins, and/or by otherabnormal physiological states, such as hyperglycemia or hypoglycemia.

In animal models of many such conditions, stimulating expression ofinducible heme oxygenase (HO-1), a target gene of the Nrf2 pathway, hasbeen shown to have a significant therapeutic effect including in modelsof myocardial infarction, renal failure, transplant failure andrejection, stroke, cardiovascular disease, and autoimmune disease (e.g.,Sacerdoti et al., 2005; Abraham & Kappas, 2005; Bach, 2006; Araujo etal., 2003; Liu et al., 2006; Ishikawa et al., 2001; Kruger et al., 2006;Satoh et al., 2006; Zhou et al., 2005; Morse and Choi, 2005; Morse andChoi, 2002). This enzyme breaks free heme down into iron, carbonmonoxide (CO), and biliverdin (which is subsequently converted to thepotent antioxidant molecule, bilirubin).

In another aspect, RTA 408 may be used in preventing or treating tissuedamage or organ failure, acute and chronic, resulting from oxidativestress exacerbated by inflammation. Examples of diseases that fall inthis category include heart failure, liver failure, transplant failureand rejection, renal failure, pancreatitis, fibrotic lung diseases(cystic fibrosis, COPD, and idiopathic pulmonary fibrosis, amongothers), diabetes (including complications), atherosclerosis,ischemia-reperfusion injury, glaucoma, stroke, autoimmune disease,autism, macular degeneration, and muscular dystrophy. For example, inthe case of autism, studies suggest that increased oxidative stress inthe central nervous system may contribute to the development of thedisease (Chauhan and Chauhan, 2006).

Evidence also links oxidative stress and inflammation to the developmentand pathology of many other disorders of the central nervous system,including psychiatric disorders, such as psychosis, major depression,and bipolar disorder; seizure disorders, such as epilepsy; pain andsensory syndromes, such as migraine, neuropathic pain, or tinnitus; andbehavioral syndromes, such as the attention deficit disorders. See,e.g., Dickerson et al., 2007; Hanson et al., 2005; Kendall-Tackett,2007; Lencz et al., 2007; Dudhgaonkar et al., 2006; Lee et al., 2007;Morris et al., 2002; Ruster et al., 2005; McIver et al., 2005;Sarchielli et al., 2006; Kawakami et al., 2006; Ross et al., 2003, whichare all incorporated by reference herein. For example, elevated levelsof inflammatory cytokines, including TNF-α, interferon-γ, and IL-6, areassociated with major mental illness (Dickerson et al., 2007).Microglial activation has also been linked to major mental illness.Therefore, downregulating inflammatory cytokines and inhibitingexcessive activation of microglia could be beneficial in patients withschizophrenia, major depression, bipolar disorder, autism-spectrumdisorders, and other neuropsychiatric disorders.

Accordingly, in pathologies involving oxidative stress alone oroxidative stress exacerbated by inflammation, treatment may compriseadministering to a subject a therapeutically effective amount of acompound of this invention, such as those described above or throughoutthis specification. Treatment may be administered preventively, inadvance of a predictable state of oxidative stress (e.g., organtransplantation or the administration of radiation therapy to a cancerpatient), or it may be administered therapeutically in settingsinvolving established oxidative stress and inflammation. In someembodiments, when a compound of the present invention is used fortreating a patient receiving radiation therapy and/or chemotherapy, thecompound of the invention may be administered before, at the same time,and/or after the radiation or chemotherapy, or the compound may beadministered in combination with the other therapies. In someembodiments, the compound of the invention may prevent and/or reduce theseverity of side effects associated with the radiation therapy orchemotherapy (using a different agent) without reducing the anticancereffects of the radiation therapy or chemotherapy. Because such sideeffects may be dose-limiting for the radiation therapy and/orchemotherapy, in some embodiments, the compound of the present inventionmay be used to allow for higher and/or more frequent dosing of theradiation therapy and/or chemotherapy, for example, resulting in greatertreatment efficacy. In some embodiments, the compound of the inventionwhen administered in combination with the radiation therapy and/orchemotherapy may enhance the efficacy of a given dose of radiationand/or chemotherapy. In some embodiments, the compound of the inventionwhen administered in combination with the radiation therapy and/orchemotherapy may enhance the efficacy of a given dose of radiationand/or chemotherapy and reduce (or, at a minimum, not add to) the sideeffects of the radiation and/or chemotherapy. In some embodiments, andwithout being bound by theory, this combinatorial efficacy may resultfrom inhibition of the activity of the pro-inflammatory transcriptionfactor NF-κB by the compound of the invention. NF-κB is oftenchronically activated in cancer cells, and such activation is associatedwith resistance to therapy and promotion of tumor progression (e.g.,Karin, 2006; Aghajan et al., 2012). Other transcription factors thatpromote inflammation and cancer, such as STAT3 (e.g., He and Karin 2011;Grivennikov and Karin, 2010), may also be inhibited by the compound ofthe invention in some embodiments.

RTA 408 may be used to treat or prevent inflammatory conditions, such assepsis, dermatitis, autoimmune disease, and osteoarthritis. RTA 408 mayalso be used to treat or prevent inflammatory pain and/or neuropathicpain, for example, by inducing Nrf2 and/or inhibiting NF-κB.

RTA 408 may also be used to treat or prevent diseases, such as cancer,inflammation, Alzheimer's disease, Parkinson's disease, multiplesclerosis, autism, amyotrophic lateral sclerosis, Huntington's disease,autoimmune diseases, such as rheumatoid arthritis, lupus, Crohn'sdisease, and psoriasis, inflammatory bowel disease, all other diseaseswhose pathogenesis is believed to involve excessive production of eithernitric oxide or prostaglandins, and pathologies involving oxidativestress alone or oxidative stress exacerbated by inflammation. RTA 408may be used in the treatment or prevention of cancers include acarcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiplemyeloma, or seminoma, or cancer of the bladder, blood, bone, brain,breast, central nervous system, cervix, colon, endometrium, esophagus,gall bladder, genitalia, genitourinary tract, head, kidney, larynx,liver, lung, muscle tissue, neck, oral or nasal mucosa, ovary, pancreas,prostate, skin, spleen, small intestine, large intestine, stomach,testicle, or thyroid.

Another aspect of inflammation is the production of inflammatoryprostaglandins, such as prostaglandin E. RTA 408 may be used to promotevasodilation, plasma extravasation, localized pain, elevatedtemperature, and other symptoms of inflammation. The inducible form ofthe enzyme COX-2 is associated with their production, and high levels ofCOX-2 are found in inflamed tissues. Consequently, inhibition of COX-2may relieve many symptoms of inflammation and a number of importantanti-inflammatory drugs (e.g., ibuprofen and celecoxib) act byinhibiting COX-2 activity. It has been demonstrated that a class ofcyclopentenone prostaglandins (cyPGs) (e.g., 15-deoxy prostaglandin J2,a.k.a. PGJ2) plays a role in stimulating the orchestrated resolution ofinflammation (e.g., Rajakariar et al., 2007). COX-2 is also associatedwith the production of cyclopentenone prostaglandins. Consequently,inhibition of COX-2 may interfere with the full resolution ofinflammation, potentially promoting the persistence of activated immunecells in tissues and leading to chronic, “smoldering” inflammation. Thiseffect may be responsible for the increased incidence of cardiovasculardisease in patients using selective COX-2 inhibitors for long periods oftime.

In one aspect, RTA 408 may be used to control the production ofpro-inflammatory cytokines within the cell by selectively activatingregulatory cysteine residues (RCRs) on proteins that regulate theactivity of redox-sensitive transcription factors. Activation of RCRs bycyPGs has been shown to initiate a pro-resolution program in which theactivity of the antioxidant and cytoprotective transcription factor Nrf2is potently induced and the activities of the pro-oxidant andpro-inflammatory transcription factors NF-κB and the STATs aresuppressed. In some embodiments, RTA 408 may be used to increase theproduction of antioxidant and reductive molecules (NQO1, HO-1, SOD1,γ-GCS) and decrease oxidative stress and the production of pro-oxidantand pro-inflammatory molecules (iNOS, COX-2, TNF-α). In someembodiments, RTA 408 may be used to cause the cells that host theinflammatory event to revert to a non-inflammatory state by promotingthe resolution of inflammation and limiting excessive tissue damage tothe host.

A. Cancer

In some embodiments, RTA 408, the polymorphic forms, and methods of thepresent disclosure may be used to induce apoptosis in tumor cells, toinduce cell differentiation, to inhibit cancer cell proliferation, toinhibit an inflammatory response, and/or to function in achemopreventative capacity. For example, the invention provides newpolymorphic forms that have one or more of the following properties: (1)an ability to induce apoptosis and differentiate both malignant andnon-malignant cells, (2) an activity at sub-micromolar or nanomolarlevels as an inhibitor of proliferation of many malignant orpremalignant cells, (3) an ability to suppress the de novo synthesis ofthe inflammatory enzyme inducible nitric oxide synthase (iNOS), (4) anability to inhibit NF-κB activation, and (5) an ability to induce theexpression of heme oxygenase-1 (HO-1).

The levels of iNOS and COX-2 are elevated in certain cancers and havebeen implicated in carcinogenesis and COX-2 inhibitors have been shownto reduce the incidence of primary colonic adenomas in humans (Rostom etal., 2007; Brown and DuBois, 2005; Crowel et al., 2003). iNOS isexpressed in myeloid-derived suppressor cells (MDSCs) (Angulo et al.,2000) and COX-2 activity in cancer cells has been shown to result in theproduction of prostaglandin E₂ (PGE₂), which has been shown to inducethe expression of arginase in MDSCs (Sinha et al., 2007). Arginase andiNOS are enzymes that utilize L-arginine as a substrate and produceL-ornithine and urea, and L-citrulline and NO, respectively. Thedepletion of arginine from the tumor microenvironment by MDSCs, combinedwith the production of NO and peroxynitrite has been shown to inhibitproliferation and induce apoptosis of T cells (Bronte et al., 2003)Inhibition of COX-2 and iNOS has been shown to reduce the accumulationof MDSCs, restore cytotoxic activity of tumor-associated T cells, anddelay tumor growth (Sinha et al., 2007; Mazzoni et al., 2002; Zhou etal., 2007).

Inhibition of the NF-κB and JAK/STAT signaling pathways has beenimplicated as a strategy to inhibit proliferation of cancer epithelialcells and induce their apoptosis. Activation of STAT3 and NF-κB has beenshown to result in suppression of apoptosis in cancer cells, andpromotion of proliferation, invasion, and metastasis. Many of the targetgenes involved in these processes have been shown to betranscriptionally regulated by both NF-κB and STAT3 (Yu et al., 2007).

In addition to their direct roles in cancer epithelial cells, NF-κB andSTAT3 also have important roles in other cells found within the tumormicroenvironment. Experiments in animal models have demonstrated thatNF-κB is required in both cancer cells and hematopoeitic cells topropagate the effects of inflammation on cancer initiation andprogression (Greten et al., 2004). NF-κB inhibition in cancer andmyeloid cells reduces the number and size, respectively, of theresultant tumors. Activation of STAT3 in cancer cells results in theproduction of several cytokines (IL-6, IL-10) which suppress thematuration of tumor-associated dendritic cells (DC). Furthermore, STAT3is activated by these cytokines in the dendritic cells themselves.Inhibition of STAT3 in mouse models of cancer restores DC maturation,promotes antitumor immunity, and inhibits tumor growth (Kortylewski etal., 2005). In some embodiments, RTA 408 and its polymorphic forms canbe used to treat cancer, including, for example, prostate cancer. Insome embodiments, RTA 408 and its polymorphic forms can be used in acombination therapy to treat cancer including, for example, prostatecancer. See, e.g., Example H below.

B. Multiple Sclerosis and Other Neurodegenerative Conditions

In some embodiments, RTA 408, the polymorphic forms, and the methods ofthis invention may be used for treating patients for multiple sclerosis(MS) or other neurodegenerative conditions such as Alzheimer's disease,Parkinson's disease, or amyotrophic lateral sclerosis. MS is known to bean inflammatory condition of the central nervous system (Williams etal., 1994; Merrill and Benvenist, 1996; Genain and Nauser, 1997). Basedon several investigations, evidence suggests that inflammatory,oxidative, and/or immune mechanisms are involved in the pathogenesis ofAlzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateralsclerosis (ALS), and MS (Bagasra et al., 1995; McGeer and McGeer, 1995;Simonian and Coyle, 1996; Kaltschmidt et al., 1997). Epidemiologic dataindicate that chronic use of NSAIDs which block synthesis ofprostaglandins from arachidonate, markedly lowers the risk fordevelopment of AD (McGeer et al., 1996; Stewart et al., 1997). Thus,agents that block formation of NO and prostaglandins, may be used inapproaches to prevent and treat neurodegenerative diseases. Successfultherapeutic candidates for treating such a disease typically require anability to penetrate the blood-brain barrier. See, for example, U.S.Patent Publication 2009/0060873, which is incorporated by referenceherein.

C. Neuroinflammation

In some embodiments, RTA 408, the polymorphic forms, and methods of thisinvention may be used for treating patients with neuroinflammation.Neuroinflammation encapsulates the idea that microglial and astrocyticresponses and actions in the central nervous system have a fundamentallyinflammation-like character, and that these responses are central to thepathogenesis and progression of a wide variety of neurologicaldisorders. These ideas have been extended from Alzheimer's disease toother neurodegenerative diseases (Eikelenboom et al., 2002; Ishizawa andDickson, 2001), to ischemic/toxic diseases (Gehrmann et al., 1995;Touzani et al., 1999), to tumor biology (Graeber et al., 2002) and evento normal brain development. Neuroinflammation incorporates a widespectrum of complex cellular responses that include activation ofmicroglia and astrocytes and induction of cytokines, chemokines,complement proteins, acute phase proteins, oxidative injury, and relatedmolecular processes, and the events may have detrimental effects onneuronal function, leading to neuronal injury, further glial activation,and ultimately neurodegeneration.

D. Renal Diseases

In some embodiments, RTA 408, as well as polymorphic forms thereof, maybe used for treating patients with renal diseases and disorders,including renal failure and chronic kidney disease (CKD), based, forexample, on the methods taught by U.S. Pat. No. 8,129,429, which isincorporated by reference herein. Renal failure, resulting in inadequateclearance of metabolic waste products from the blood and abnormalconcentrations of electrolytes in the blood, is a significant medicalproblem throughout the world, especially in developed countries.Diabetes and hypertension are among the most important causes of chronicrenal failure, also known as chronic kidney disease (CKD), but it isalso associated with other conditions such as lupus. Acute renal failuremay arise from exposure to certain drugs (e.g., acetaminophen) or toxicchemicals, or from ischemia-reperfusion injury associated with shock orsurgical procedures such as transplantation, and may result in chronicrenal failure. In many patients, renal failure advances to a stage inwhich the patient requires regular dialysis or kidney transplantation tocontinue living. Both of these procedures are highly invasive andassociated with significant side effects and quality of life issues.Although there are effective treatments for some complications of renalfailure, such as hyperparathyroidism and hyperphosphatemia, no availabletreatment has been shown to halt or reverse the underlying progressionof renal failure. Thus, agents that can improve compromised renalfunction would represent a significant advance in the treatment of renalfailure.

Inflammation contributes significantly to the pathology of CKD. There isalso a strong mechanistic link between oxidative stress and renaldysfunction. The NF-κB signaling pathway plays an important role in theprogression of CKD as NF-κB regulates the transcription of MCP-1, achemokine that is responsible for the recruitment ofmonocytes/macrophages resulting in an inflammatory response thatultimately injures the kidney (Wardle, 2001). The Keap1/Nrf2/ARE pathwaycontrols the transcription of several genes encoding antioxidantenzymes, including heme oxygenase-1 (HO-1). Ablation of the Nrf2 gene infemale mice results in the development of lupus-like glomerularnephritis (Yoh et al., 2001). Furthermore, several studies havedemonstrated that HO-1 expression is induced in response to renal damageand inflammation and that this enzyme and its products—bilirubin andcarbon monoxide—play a protective role in the kidney (Nath et al.,2006).

Acute kidney injury (AKI) can occur following ischemia-reperfusion,treatment with certain pharmacological agents, such as cisplatin andrapamycin, and intravenous injection of radiocontrast media used inmedical imaging. As in CKD, inflammation and oxidative stress contributeto the pathology of AKI. The molecular mechanisms underlyingradiocontrast-induced nephropathy (RCN) are not well understood;however, it is likely that a combination of events including prolongedvasoconstriction, impaired kidney autoregulation, and direct toxicity ofthe contrast media all contribute to renal failure (Tumlin et al.,2006). Vasoconstriction results in decreased renal blood flow and causesischemia-reperfusion and the production of reactive oxygen species. HO-1is strongly induced under these conditions and has been demonstrated toprevent ischemia-reperfusion injury in several different organs,including the kidney (Nath et al., 2006). Specifically, induction ofHO-1 has been shown to be protective in a rat model of RCN (Goodman etal., 2007). Reperfusion also induces an inflammatory response, in partthough activation of NF-κB signaling (Nichols, 2004). Targeting NF-κBhas been proposed as a therapeutic strategy to prevent organ damage(Zingarelli et al., 2003).

E. Cardiovascular Disease

In some embodiments, RTA 408, the polymorphic forms and methods of thisinvention may be used for treating patients with cardiovascular disease.The etiology of CV disease is complex, but the majority of causes arerelated to inadequate or completely disrupted supply of blood to acritical organ or tissue. Frequently such a condition arises from therupture of one or more atherosclerotic plaques, which leads to theformation of a thrombus that blocks blood flow in a critical vessel.

In some incidences, atherosclerosis may be so extensive in criticalblood vessels that stenosis (narrowing of the arteries) develops andblood flow to critical organs (including the heart) is chronicallyinsufficient. Such chronic ischemia can lead to end-organ damage of manykinds, including the cardiac hypertrophy associated with congestiveheart failure.

Atherosclerosis, the underlying defect leading to many forms ofcardiovascular disease, occurs when a physical defect or injury to thelining (endothelium) of an artery triggers an inflammatory responseinvolving the proliferation of vascular smooth muscle cells and theinfiltration of leukocytes into the affected area. Ultimately, acomplicated lesion known as an atherosclerotic plaque may form, composedof the above-mentioned cells combined with deposits ofcholesterol-bearing lipoproteins and other materials (e.g., Hansson etal., 2006). Despite the significant benefits offered by currenttherapeutic treatments, mortality from cardiovascular disease remainshigh and significant unmet needs in the treatment of cardiovasculardisease remain.

Induction of HO-1 has been shown to be beneficial in a variety of modelsof cardiovascular disease, and low levels of HO-1 expression have beenclinically correlated with elevated risk of CV disease. RTA 408, thepolymorphic forms and methods of the invention, therefore, may be usedin treating or preventing a variety of cardiovascular disordersincluding but not limited to atherosclerosis, hypertension, myocardialinfarction, chronic heart failure, stroke, subarachnoid hemorrhage, andrestenosis. In some embodiments, RTA 408, the polymorphic forms andmethods of the invention may be used as a combination therapy with otherknown cardiovascular therapies such as but not limited toanticoagulants, thrombolytics, streptokinase, tissue plasminogenactivators, surgery, coronary artery bypass grafting, balloonangioplasty, the use of stents, drugs which inhibit cell proliferation,or drugs which lower cholesterol levels.

F. Diabetes

In some embodiments, RTA 408, as well as polymorphic forms thereof, maybe used for treating patients with diabetes, based, for example, on themethods taught by U.S. Pat. No. 8,129,429, which is incorporated byreference herein. Diabetes is a complex disease characterized by thebody's failure to regulate circulating levels of glucose. This failuremay result from a lack of insulin, a peptide hormone that regulates boththe production and absorption of glucose in various tissues. Deficientinsulin compromises the ability of muscle, fat, and other tissues toabsorb glucose properly, leading to hyperglycemia (abnormally highlevels of glucose in the blood). Most commonly, such insulin deficiencyresults from inadequate production in the islet cells of the pancreas.In the majority of cases this arises from autoimmune destruction ofthese cells, a condition known as type 1 or juvenile-onset diabetes, butmay also be due to physical trauma or some other cause.

Diabetes may also arise when muscle and fat cells become less responsiveto insulin and do not absorb glucose properly, resulting inhyperglycemia. This phenomenon is known as insulin resistance, and theresulting condition is known as type 2 diabetes. Type 2 diabetes, themost common type, is highly associated with obesity and hypertension.Obesity is associated with an inflammatory state of adipose tissue thatis thought to play a major role in the development of insulin resistance(e.g., Hotamisligil, 2006; Guilherme et al., 2008).

Diabetes is associated with damage to many tissues, largely becausehyperglycemia (and hypoglycemia, which can result from excessive orpoorly timed doses of insulin) is a significant source of oxidativestress. Because of their ability to protect against oxidative stress,particularly by the induction of HO-1 expression, RTA 408, thepolymorphic forms, and methods of the current invention may be used intreatments for many complications of diabetes. As noted above (Cai etal., 2005), chronic inflammation and oxidative stress in the liver aresuspected to be primary contributing factors in the development of type2 diabetes. Furthermore, PPAR_(γ) agonists such as thiazolidinedionesare capable of reducing insulin resistance and are known to be effectivetreatments for type 2 diabetes. In some embodiments, RTA 408, thepolymorphic forms, and methods of the current invention may be used ascombination therapies with PPAR_(γ) agonists such as thiazolidinediones.

G. Arthritis

In some embodiments, RTA 408, the polymorphic forms, and methods of thisinvention may be used for treating patients with a form of arthritis. Insome embodiments, the forms of arthritis that could be treated with RTA408 and the polymorphic forms of this invention are rheumatoid arthritis(RA), psoriatic arthritis (PsA), spondyloarthropathies (SpAs) includingankylosing spondylitis (AS), reactive arthritis (ReA), and enteropathicarthritis (EA), juvenile rheumatoid arthritis (JRA), and earlyinflammatory arthritis.

For rheumatoid arthritis, the first signs typically appear in thesynovial lining layer, with proliferation of synovial fibroblasts andtheir attachment to the articular surface at the joint margin (Lipsky,1998). Subsequently, macrophages, T cells and other inflammatory cellsare recruited into the joint, where they produce a number of mediators,including the cytokines interleukin-1 (IL-1), which contributes to thechronic sequelae leading to bone and cartilage destruction, and tumornecrosis factor (TNF-α), which plays a role in inflammation (Dinarello,1998; Arend and Dayer, 1995; van den Berg, 2001). The concentration ofIL-1 in plasma is significantly higher in patients with RA than inhealthy individuals and, notably, plasma IL-1 levels correlate with RAdisease activity (Eastgate et al., 1988). Moreover, synovial fluidlevels of IL-1 are correlated with various radiographic and histologicfeatures of RA (Kahle et al., 1992; Rooney et al., 1990).

Other forms of arthritis include psoriatic arthritis (PsA), which is achronic inflammatory arthropathy characterized by the association ofarthritis and psoriasis. Studies have revealed that PsA shares a numberof genetic, pathogenic and clinical features with otherspondyloarthropathies (SpAs), a group of diseases that compriseankylosing spondylitis, reactive arthritis and enteropathic arthritis(Wright, 1979). The notion that PsA belongs to the SpA group hasrecently gained further support from imaging studies demonstratingwidespread enthesitis in PsA but not RA (McGonagle et al., 1999;McGonagle et al., 1998). More specifically, enthesitis has beenpostulated to be one of the earliest events occurring in the SpAs,leading to bone remodeling and ankylosis in the spine, as well as toarticular synovitis when the inflamed entheses are close to peripheraljoints. Increased amounts of TNF-α have been reported in both psoriaticskin (Ettehadi et al., 1994) and synovial fluid (Partsch et al., 1997).Recent trials have shown a positive benefit of anti-TNF treatment inboth PsA (Mease et al., 2000) and ankylosing spondylitis (Brandt et al.,2000).

Juvenile rheumatoid arthritis (JRA), a term for the most prevalent formof arthritis in children, is applied to a family of illnessescharacterized by chronic inflammation and hypertrophy of the synovialmembranes. The term overlaps, but is not completely synonymous, with thefamily of illnesses referred to as juvenile chronic arthritis and/orjuvenile idiopathic arthritis in Europe.

Polyarticular JRA is a distinct clinical subtype characterized byinflammation and synovial proliferation in multiple joints (four ormore), including the small joints of the hands (Jarvis, 2002). Thissubtype of JRA may be severe, because of both its multiple jointinvolvement and its capacity to progress rapidly over time. Althoughclinically distinct, polyarticular JRA is not homogeneous, and patientsvary in disease manifestations, age of onset, prognosis, and therapeuticresponse. These differences very likely reflect a spectrum of variationin the nature of the immune and inflammatory attack that can occur inthis disease (Jarvis, 1998).

Ankylosing spondylitis (AS) is a disease subset within a broader diseaseclassification of spondyloarthropathy. Patients affected with thevarious subsets of spondyloarthropathy have disease etiologies that areoften very different, ranging from bacterial infections to inheritance.Yet, in all subgroups, the end result of the disease process is axialarthritis.

AS is a chronic systemic inflammatory rheumatic disorder of the axialskeleton with or without extraskeletal manifestations. Sacroiliac jointsand the spine are primarily affected, but hip and shoulder joints, andless commonly peripheral joints or certain extra-articular structuressuch as the eye, vasculature, nervous system, and gastrointestinalsystem may also be involved. The disease's etiology is not yet fullyunderstood (Wordsworth, 1995; Calin and Taurog, 1998). The etiology isstrongly associated with the major histocompatibility class I (MHC I)HLA-B27 allele (Calin and Taurog, 1998). AS affects individuals in theprime of their life and is feared because of its potential to causechronic pain and irreversible damage of tendons, ligaments, joints, andbones (Brewerton et al., 1973a; Brewerton et al., 1973b; Schlosstein etal., 1973).

H. Ulcerative Colitis

In some embodiments, RTA 408, the polymorphic forms and methods of thisinvention may be used for treating patients with ulcerative colitis.Ulcerative colitis is a disease that causes inflammation and sores,called ulcers, in the lining of the large intestine. The inflammationusually occurs in the rectum and lower part of the colon, but it mayaffect the entire colon. Ulcerative colitis may also be called colitisor proctitis. The inflammation makes the colon empty frequently, causingdiarrhea. Ulcers form in places where the inflammation has killed thecells lining the colon and the ulcers bleed and produce pus.

Ulcerative colitis is an inflammatory bowel disease (IBD), the generalname for diseases that cause inflammation in the small intestine andcolon. Ulcerative colitis can be difficult to diagnose because itssymptoms are similar to other intestinal disorders and to another typeof IBD, Crohn's disease. Crohn's disease differs from ulcerative colitisbecause it causes inflammation deeper within the intestinal wall. Also,Crohn's disease usually occurs in the small intestine, although thedisease can also occur in the mouth, esophagus, stomach, duodenum, largeintestine, appendix, and anus.

I. Crohn's Disease

In some embodiments, RTA 408, the polymorphic forms, and methods of thisinvention may be used for treating patients with Crohn's disease.Crohn's disease symptoms include intestinal inflammation and thedevelopment of intestinal stenosis and fistulas; neuropathy oftenaccompanies these symptoms. Anti-inflammatory drugs, such as5-aminosalicylates (e.g., mesalamine) or corticosteroids, are typicallyprescribed, but are not always effective (reviewed in Botoman et al.,1998). Immunosuppression with cyclosporine is sometimes beneficial forpatients resistant to or intolerant of corticosteroids (Brynskov et al.,1989).

In active cases of Crohn's disease, elevated concentrations of TNF-α andIL-6 are secreted into the blood circulation, and TNF-α, IL-1, IL-6, andIL-8 are produced in excess locally by mucosal cells (id.; Funakoshi etal., 1998). These cytokines can have far-ranging effects onphysiological systems including bone development, hematopoiesis, andliver, thyroid, and neuropsychiatric function. Also, an imbalance of theIL-1β/IL-1ra ratio, in favor of pro-inflammatory IL-1β, has beenobserved in patients with Crohn's disease (Rogler and Andus, 1998; Saikiet al., 1998; Dionne et al., 1998; but see Kuboyama, 1998).

Treatments that have been proposed for Crohn's disease include the useof various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., ofIL-1β converting enzyme and antioxidants) and anti-cytokine antibodies(Rogler and Andus, 1998; van Hogezand and Verspaget, 1998; Reimund etal., 1998; Lugering et al., 1998; McAlindon et al., 1998). Inparticular, monoclonal antibodies against TNF-α have been tried withsome success in the treatment of Crohn's disease (Targan et al., 1997;Stack et al., 1997; van Dullemen et al., 1995). These compounds may beused in combination therapy with RTA 408, the polymorphic forms, andmethods of the present disclosure.

J. Systemic Lupus Erythematosus

In some embodiments, RTA 408, the polymorphic forms and methods of thisinvention may be used for treating patients with SLE. Systemic lupuserythematosus (SLE) is an autoimmune rheumatic disease characterized bydeposition in tissues of autoantibodies and immune complexes leading totissue injury (Kotzin, 1996). In contrast to autoimmune diseases, suchas MS and type 1 diabetes mellitus, SLE potentially involves multipleorgan systems directly, and its clinical manifestations are diverse andvariable (reviewed by Kotzin and O'Dell, 1995). For example, somepatients may demonstrate primarily skin rash and joint pain, showspontaneous remissions, and require little medication. At the other endof the spectrum are patients who demonstrate severe and progressivekidney involvement that requires therapy with high doses of steroids andcytotoxic drugs such as cyclophosphamide (Kotzin, 1996).

One of the antibodies produced by SLE, IgG anti-dsDNA, plays a majorrole in the development of lupus glomerulonephritis (GN) (Hahn and Tsao,1993; Ohnishi et al., 1994). Glomerulonephritis is a serious conditionin which the capillary walls of the kidney's blood purifying glomerulibecome thickened by accretions on the epithelial side of glomerularbasement membranes. The disease is often chronic and progressive and maylead to eventual renal failure.

K. Irritable Bowel Syndrome

In some embodiments, RTA 408, the polymorphic forms, and methods of thisinvention may be used for treating patients with irritable bowelsyndrome (IBS). IBS is a functional disorder characterized by abdominalpain and altered bowel habits. This syndrome may begin in youngadulthood and can be associated with significant disability. Thissyndrome is not a homogeneous disorder. Rather, subtypes of IBS havebeen described on the basis of the predominant symptom—diarrhea,constipation, or pain. In the absence of “alarm” symptoms, such asfever, weight loss, and gastrointestinal bleeding, a limited workup isneeded.

Increasingly, evidence for the origins of IBS suggests a relationshipbetween infectious enteritis and subsequent development of IBS.Inflammatory cytokines may play a role. In a survey of patients with ahistory of confirmed bacterial gastroenteritis (Neal et al., 1997), 25%reported persistent alteration of bowel habits. Persistence of symptomsmay be due to psychological stress at the time of acute infection (Gweeet al., 1999).

Recent data suggest that bacterial overgrowth in the small intestine mayalso have a role in IBS symptoms. In one study (Pimentel et al., 2000),157 (78%) of 202 IBS patients referred for hydrogen breath testing hadtest findings that were positive for bacterial overgrowth. Of the 47subjects who had follow-up testing, 25 (53%) reported improvement insymptoms (i.e., abdominal pain and diarrhea) with antibiotic treatment.

L. Sjögren's Syndrome

In some embodiments, RTA 408, the polymorphic forms, and methods of thisinvention may be used for treating patients with Sjögren's syndrome.Primary Sjögren's syndrome (SS) is a chronic, slowly progressive,systemic autoimmune disease, which affects predominantly middle-agedwomen (female-to-male ratio 9:1), although it can be seen in all agesincluding childhood (Jonsson et al., 2002). The disease is characterizedby lymphocytic infiltration and destruction of the exocrine glands,which are infiltrated by mononuclear cells including CD4+, CD8+lymphocytes, and B-cells (Jonsson et al., 2002). In addition,extraglandular (systemic) manifestations are seen in one-third ofpatients (Jonsson et al., 2001).

In other systemic autoimmune diseases, such as RA, factors critical forectopic germinal centers (GCs) have been identified. Rheumatoid synovialtissues with GCs were shown to produce chemokines CXCL13, CCL21, andlymphotoxin (LT)-β (detected on follicular center and mantle zone Bcells). Multivariate regression analysis of these analytes identifiedCXCL13 and LT-β as the solitary cytokines predicting GCs in rheumatoidsynovitis (Weyand and Goronzy, 2003). Recently CXCL13 and CXCR5 insalivary glands has been shown to play an essential role in theinflammatory process by recruiting B and T cells, therefore contributingto lymphoid neogenesis and ectopic GC formation in SS (Salomonsson etal., 2002).

M. Psoriasis

In some embodiments, RTA 408, the polymorphic forms, and methods of thisinvention may be used for treating patients with psoriasis. Psoriasis isa chronic skin disease of scaling and inflammation that affects 2 to 2.6percent of the United States population, or between 5.8 and 7.5 millionpeople. Psoriasis occurs when skin cells quickly rise from their originbelow the surface of the skin and pile up on the surface before theyhave a chance to mature. Usually this movement (also called turnover)takes about a month, but in psoriasis turnover may occur in only a fewdays. In its typical form, psoriasis results in patches of thick, red(inflamed) skin covered with silvery scales. These patches, which aresometimes referred to as plaques, usually itch or feel sore. The plaquesmost often occur on the elbows, knees, other parts of the legs, scalp,lower back, face, palms, and soles of the feet, but they can occur onskin anywhere on the body. The disease may also affect the fingernails,the toenails, and the soft tissues of the genitals and inside the mouth.

Psoriasis is a skin disorder driven by the immune system, especiallyinvolving a type of white blood cell called a T cell. Normally, T cellshelp protect the body against infection and disease. In the case ofpsoriasis, T cells are put into action by mistake and become so activethat they trigger other immune responses, which lead to inflammation andto rapid turnover of skin cells.

N. Infectious diseases

In some embodiments, RTA 408, the polymorphic forms, and methods of thepresent disclosure may be useful in the treatment of infectiousdiseases, including viral and bacterial infections. As noted above, suchinfections may be associated with severe localized or systemicinflammatory responses. For example, influenza may cause severeinflammation of the lung and bacterial infection can cause the systemichyperinflammatory response, including the excessive production ofmultiple inflammatory cytokines, which is the hallmark of sepsis. Inaddition, compounds of the invention may be useful in directlyinhibiting the replication of viral pathogens. Previous studies havedemonstrated that related compounds such as CDDO can inhibit thereplication of HIV in macrophages (Vazquez et al., 2005). Other studieshave indicated that inhibition of NF-κB signaling may inhibit influenzavirus replication, and that cyclopentenone prostaglandins may inhibitviral replication (e.g., Mazur et al., 2007; Pica et al., 2000).

The present invention relates to the treatment or prevention of each ofthe diseases/disorders/conditions referred to above in section IV. usingthe compound RTA 408 or a pharmaceutically acceptable salt thereof, or apolymorphic form of that compound (such as, e.g., any one of thepolymorphic forms described herein above or below), or a pharmaceuticalcomposition comprising any of the aforementioned entities and apharmaceutically acceptable carrier (including, e.g., the pharmaceuticalcompositions described above).

V. Pharmaceutical Formulations and Routes of Administration

RTA 408 may be administered by a variety of methods, e.g., orally or byinjection (e.g., subcutaneous, intravenous, intraperitoneal, etc.).Depending on the route of administration, the active compounds may becoated in a material to protect the compound from the action of acidsand other natural conditions which may inactivate the compound. They mayalso be administered by continuous perfusion/infusion of a disease orwound site.

To administer RTA 408 by other than parenteral administration, it may benecessary to coat the compound with, or co-administer the compound with,a material to prevent its inactivation. For example, the therapeuticcompound may be administered to a patient in an appropriate carrier, forexample, liposomes, or a diluent. Pharmaceutically acceptable diluentsinclude saline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al., 1984).

RTA 408 may also be administered parenterally, intraperitoneally,intraspinally, or intracerebrally. Dispersions can be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.

Sterile injectable solutions can be prepared by incorporating RTA 408 inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thetherapeutic compound into a sterile carrier that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yields a powder of the activeingredient (i.e., the therapeutic compound) plus any additional desiredingredient from a previously sterile-filtered solution thereof.

RTA 408 may be rendered fully amorphous using a direct spray dryingprocedure. RTA 408 can be orally administered, for example, with aninert diluent or an assimilable edible carrier. The therapeutic compoundand other ingredients may also be enclosed in a hard or soft shellgelatin capsule, compressed into tablets, or incorporated directly intothe patient's diet. For oral therapeutic administration, the therapeuticcompound may be incorporated, for example, with excipients and used inthe form of ingestible tablets, buccal tablets, troches, capsulesincluding hard or soft capsules, elixirs, emulsions, solid dispersions,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the patients to be treated, each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient.

RTA 408 may also be administered topically to the skin, eye, or mucosa.In some embodiments, the compound may be prepared in a lotion, cream,gel, oil, ointment, salve, solution, suspension, or emulsion.Alternatively, if local delivery to the lungs is desired the therapeuticcompound may be administered by inhalation in a dry-powder or aerosolformulation.

RTA 408 will typically be administered at a therapeutically effectivedosage sufficient to treat a condition associated with a given patient.For example, the efficacy of a compound can be evaluated in an animalmodel system that may be predictive of efficacy in treating the diseasein humans, such as the model systems shown in the examples and drawings.

The actual dosage amount of RTA 408 or composition comprising RTA 408administered to a patient may be determined by physical andphysiological factors, such as age, sex, body weight, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient, and the route ofadministration. These factors may be determined by a skilled artisan.The practitioner responsible for administration will typically determinethe concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual patient. The dosage may beadjusted by the individual physician in the event of any complication.

An effective amount typically will vary from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, fromabout 10.0 mg/kg to about 150 mg/kg in one or more dose administrationsdaily, for one or several days (depending of course of the mode ofadministration and the factors discussed above). Other suitable doseranges include 1 mg to 10000 mg per day, 100 mg to 10000 mg per day, 500mg to 10000 mg per day, and 500 mg to 1000 mg per day. In someparticular embodiments, the amount is less than 10,000 mg per day with arange of 750 mg to 9000 mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 25 mg/kg/day, or less than 10 mg/kg/day. It mayalternatively be in the range of 1 mg/kg/day to 200 mg/kg/day. In someembodiments, the amount could be 10, 30, 100, or 150 mg/kg formulated asa suspension in sesame oil as described below in Example C1. In someembodiments, the amount could be 3, 10, 30 or 100 mg/kg administereddaily via oral gavage as described below in Examples C2 and C3. In someembodiments, the amount could be 10, 30, or 100 mg/kg administeredorally as described below in Example C6. For example, regardingtreatment of diabetic patients, the unit dosage may be an amount thatreduces blood glucose by at least 40% as compared to an untreatedpatient. In another embodiment, the unit dosage is an amount thatreduces blood glucose to a level that is ±10% of the blood glucose levelof a non-diabetic patient.

In other non-limiting examples, a dose may also comprise from about 1μg/kg body weight, about 5 μg/kg body weight, about 10 μg/kg bodyweight, about 50 μg/kg body weight, about 100 μg/kg body weight, about200 μg/kg body weight, about 350 μg/kg body weight, about 500 μg/kg bodyweight, about 1 mg/kg body weight, about 5 mg/kg body weight, about 10mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg bodyweight, about 200 mg/kg body weight, about 350 mg/kg body weight, about500 mg/kg body weight, to about 1000 mg/kg body weight or more peradministration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 5 mg/kg body weight to about 100 mg/kg body weight, about 5μg/kg/body weight to about 500 mg/kg body weight, etc., can beadministered, based on the numbers described above.

In certain embodiments, a pharmaceutical composition of the presentdisclosure may comprise, for example, at least about 0.01% of RTA 408.In other embodiments, RTA 408 may comprise between about 0.01% to about75% of the weight of the unit, or between about 0.01% to about 5%, forexample, and any range derivable therein. In some embodiments, RTA 408may be used in a formulation such as a suspension in sesame oil of0.01%, 0.1%, or 1% as described below in Examples F and G. In someembodiments, RTA 408 may be formulated for topical administration to theskin or eye, using a pharmaceutically suitable carrier or as asuspension, emulsion, or solution in concentrations ranging from about0.01% to 10%. In some embodiments the concentration ranges from about0.1% to about 5%. The optimal concentration may vary depending upon thetarget organ, the specific preparation, and the condition to be treated.

Single or multiple doses of the agent comprising RTA 408 arecontemplated. Desired time intervals for delivery of multiple doses canbe determined by one of ordinary skill in the art employing no more thanroutine experimentation. As an example, patients may be administered twodoses daily at approximately 12 hour intervals. In some embodiments, theagent is administered once a day. The agent(s) may be administered on aroutine schedule. As used herein a routine schedule refers to apredetermined designated period of time. The routine schedule mayencompass periods of time that are identical or that differ in length,as long as the schedule is predetermined. For instance, the routineschedule may involve administration twice a day, every day, every twodays, every three days, every four days, every five days, every sixdays, a weekly basis, a monthly basis, or any set number of days orweeks there-between. Alternatively, the predetermined routine schedulemay involve administration on a twice daily basis for the first week,followed by a daily basis for several months, etc. In other embodiments,the invention provides that the agent(s) may be taken orally and thatthe timing of which is or is not dependent upon food intake. Thus, forexample, the agent can be taken every morning and/or every evening,regardless of when the patient has eaten or will eat.

VI. Combination Therapy

In addition to being used as a monotherapy, RTA 408 and the polymorphicforms described in the present invention may also find use incombination therapies. Effective combination therapy may be achievedwith a single composition or pharmacological formulation that includesboth agents, or with two distinct compositions or formulations,administered at the same time, wherein one composition includes RTA 408or its polymorphic forms, and the other includes the second agent(s).The other therapeutic modality may be administered before, concurrentlywith, or following administration of RTA 408 or its polymorphic forms.The therapy using RTA 408 or its polymorphic forms may precede or followadministration of the other agent(s) by intervals ranging from minutesto weeks. In embodiments where the other agent and RTA 408 or itspolymorphic forms are administered separately, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that each agent would still be able to exert anadvantageously combined effect. In such instances, it is contemplatedthat one would typically administer RTA 408 or the polymorphic forms andthe other therapeutic agent within about 12-24 hours of each other and,more preferably, within about 6-12 hours of each other, with a delaytime of only about 12 hours being most preferred. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of RTA 408 orits polymorphic forms, or the other agent will be desired. In thisregard, various combinations may be employed. By way of illustration,where RTA 408 or its polymorphic forms is “A” and the other agent is“B”, the following permutations based on 3 and 4 total administrationsare exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are likewise contemplated. Non-limiting examples ofpharmacological agents that may be used in the present invention includeany pharmacological agent known to be of benefit in the treatment of acancer. In some embodiments, combinations of RTA 408 or its polymorphicforms with a cancer targeting immunotherapy, gene therapy, radiotherapy,chemotherapeutic agent, or surgery are contemplated. Also contemplatedis a combination of RTA 408 or its polymorphic forms with more than oneof the above mentioned methods including more than one type of aspecific therapy. In some embodiments, the immunotherapy can be othercancer targeting antibodies such as but not limited to trastuzumab(Herceptin®), alemtuzumab (Campath®), bevacizumab (Avastin®), cetuximab(Eribitux®), and panitumumab (Vectibix®) or conjugated antibodies suchas ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), brentuximabvedotin (Adcetris®), ado-trastuzumab emtansine (Kadcyla™), or denileukindititox (Ontak®). Furthermore, in some embodiments, RTA 408 or itspolymorphic forms are envisioned to be used in combination therapieswith dendritic cell-based immunotherapies such as Sipuleucel-T(Provenge®) or adoptive T-cell immunotherapies.

Furthermore, it is contemplated that RTA 408 or its polymorphic formsare used in combination with a chemotherapeutic agent such as but notlimited to anthracyclines, taxanes, methotrexate, mitoxantrone,estramustine, doxorubicin, etoposide, vinblastine, carboplatin,vinorelbine, 5-fluorouracil, cisplatin, topotecan, ifosfamide,cyclophosphamide, epirubicin, gemcitabine, vinorelbine, irinotecan,etoposide, vinblastine, pemetrexed, melphalan, capecitabine, andoxaliplatin. In some embodiments, RTA 408 or its polymorphic forms areused in combination with radiation therapy including but not limited tothe use of ionizing radiation. In some embodiments, the effects of thecancer therapeutic agent are synergistically enhanced throughadministration with RTA 408 and its polymorphic forms. In someembodiments, combination therapies which included RTA 408 are used totreat cancer including for example, prostate cancer. See, e.g., ExampleH below.

In some embodiments, the methods may further comprise (1) contacting atumor cell with the compound prior to contacting the tumor cell with thesecond chemotherapeutic agent, (2) contacting a tumor cell with thesecond chemotherapeutic agent prior to contacting the tumor cell withthe compound, or (3) contacting a tumor cell with the compound and thesecond chemotherapeutic agent at the same time. The secondchemotherapeutic agent may, in certain embodiments, be an antibiotic,anti-inflammatory, anti-neoplastic, anti-proliferative, anti-viral,immunomodulatory, or immunosuppressive. In other embodiments, the secondchemotherapeutic agent may be an alkylating agent, androgen receptormodulator, cytoskeletal disruptor, estrogen receptor modulator,histone-deacetylase inhibitor, HMG-CoA reductase inhibitor,prenyl-protein transferase inhibitor, retinoid receptor modulator,topoisomerase inhibitor, or tyrosine kinase inhibitor. In certainembodiments, the second chemotherapeutic agent is 5-azacitidine,5-fluorouracil, 9-cis-retinoic acid, actinomycin D, alitretinoin,all-trans-retinoic acid, annamycin, axitinib, belinostat, bevacizumab,bexarotene, bosutinib, busulfan, capecitabine, carboplatin, carmustine,CD437, cediranib, cetuximab, chlorambucil, cisplatin, cyclophosphamide,cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine, docetaxel,dolastatin-10, doxifluridine, doxorubicin, doxorubicin, epirubicin,erlotinib, etoposide, gefitinib, gemcitabine, gemtuzumab ozogamicin,hexamethylmelamine, idarubicin, ifosfamide, imatinib, irinotecan,isotretinoin, ixabepilone, lapatinib, LBH589, lomustine,mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin,mitoxantrone, MS-275, neratinib, nilotinib, nitrosourea, oxaliplatin,paclitaxel, plicamycin, procarbazine, semaxanib, semustine, sodiumbutyrate, sodium phenylacetate, streptozotocin, suberoylanilidehydroxamic acid, sunitinib, tamoxifen, teniposide, thiopeta, tioguanine,topotecan, TRAIL, trastuzumab, tretinoin, trichostatin A, valproic acid,valrubicin, vandetanib, vinblastine, vincristine, vindesine, orvinorelbine.

Additionally, combination therapies for the treatment of cardiovasculardisease utilizing RTA 408, polymorphic forms, and pharmaceuticalcompositions of the present disclosure are contemplated. For example,such methods may further comprise administering a pharmaceuticallyeffective amount of one or more cardiovascular drugs in addition to RTA408, polymorphic forms, and pharmaceutical compositions of the presentdisclosure. The cardiovascular drug may be but not limited to, forexample, a cholesterol lowering drug, an anti-hyperlipidemic, a calciumchannel blocker, an anti-hypertensive, or an HMG-CoA reductaseinhibitor. In some embodiments, non-limiting examples of cardiovasculardrugs include amlodipine, aspirin, ezetimibe, felodipine, lacidipine,lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine ornitrendipine. In other embodiments, other non-limiting examples ofcardiovascular drugs include atenolol, bucindolol, carvedilol,clonidine, doxazosin, indoramin, labetalol, methyldopa, metoprolol,nadolol, oxprenolol, phenoxybenzamine, phentolamine, pindolol, prazosin,propranolol, terazosin, timolol or tolazoline. In other embodiments, thecardiovascular drug may be, for example, a statin, such as atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin or simvastatin.

VII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

A. Synthesis of RTA 408 (63415)

Compound 1:

To a solution of toluene (400 mL), RTA 401 (which can be preparedaccording to the methods taught, for example, by Honda, et al., 1998;Honda et al., 2000b; Honda et al., 2002; Yates et al., 2007; and U.S.Pat. Nos. 6,326,507 and 6,974,801, which are incorporated herein byreference) (20.0 g, 40.6 mmol) and Et₃N (17.0 mL, 122.0 mmol) were addedinto a reactor and cooled to 0° C. with stirring. Diphenyl phosphorylazide (DPPA) (13.2 mL, 61.0 mmol) was added with stirring at 0° C. over5 min and the mixture was continually stirred at room temperatureovernight (HPLC-MS check shows no RTA 401 left). The reaction mixturewas directly loaded on a silica gel column and purified by columnchromatography (silica gel, 0% to 5% EtOAc in CH₂Cl₂) to give compound 1(19.7 g, ˜94%, partially converted into compound 2) as a white foam.

Compound 2:

Compound 1 (19.7 g, ˜38.1 mmol) and benzene (250 mL) were added into areactor and heated to 80° C. with stirring for 2 h (HPLC-MS check showsno compound 1 left). The reaction mixture was concentrated at reducedpressure to afford crude compound 2 as a solid residue, which was usedfor the next step without purification.

Compound 3:

Crude compound 2 (≦38.1 mmol) and CH₃CN (200 mL) were added into areactor and cooled to 0° C. with stirring. HCl (12 N, 90 mL) was addedat 0° C. over 1 min and the mixture was continually stirred at roomtemperature for 1 h (HPLC-MS check shows no compound 2 left). Thereaction mixture was cooled to 0° C. and 10% NaOH (˜500 mL) was addedwith stirring. Then, saturated NaHCO₃ (1 L) was added with stirring. Theaqueous phase was extracted by EtOAc (2×500 mL). The combined organicphase was washed by H₂O (200 mL), saturated NaCl (200 mL), dried overNa₂SO₄, and concentrated to afford crude compound 3 (16.62 g) as a lightyellow foam, which was used for the next step without purification.

RTA 408:

Crude amine 3 (16.62 g, 35.9 mmol), CH₃CF₂CO₂H (4.7388 g, 43.1 mmol),and CH₂Cl₂ (360 mL) were added into a reactor with stirring at roomtemperature. Then, dicyclohexylcarbodiimide (DCC) (11.129 g, 53.9 mmol)and 4-(dimethylamino)-pyridine (DMAP) (1.65 g, 13.64 mmol) were addedand the mixture was continually stirred at room temperature overnight(HPLC-MS check shows no compound 3 left). The reaction mixture wasfiltered to remove solid by-products and the filtrate was directlyloaded on a silica gel column and purified by column chromatography(silica gel, 0% to 20% EtOAc in Hexanes) twice to give compound RTA 408(16.347 g, 73% from RTA 401 over 4 steps) as a white foam: ¹H NMR (400MHz, CD₃Cl) δ 8.04 (s, 1H), 6.00 (s, 1H), 5.94 (s, br, 1H), 3.01 (d, 1H,J=4.8 Hz), 2.75-2.82 (m, 1H), 1.92-2.18 (m, 4H), 1.69-1.85 (m, 7H),1.53-1.64 (m, 1H), 1.60 (s, 3H), 1.50 (s, 3H), 1.42 (s, 3H), 1.11-1.38(m, 3H), 1.27 (s, 3H), 1.18 (s, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 0.92(s, 3H); m/z 555 (M+1).

B. Pharmacodynamics

A summary of the in vitro and in vivo studies to evaluate the primarypharmacodynamic effects of RTA 408 is provided below.

1. Effects of RTA 408 on Keap1-Nrf2 and NF-κB in Vitro

Inhibition of IFNγ-induced NO production by AIMs is Nrf2-dependent(Dinkova-Kostova, 2005). RAW264.7 mouse macrophages were plated in96-well plates at 30,000 cells/well in triplicated in RPMI 1640supplemented with 0.5% FBS and incubated at 37° C. with 5% CO₂. On thenext day, cells were pre-treated with DMSO (vehicle) or RTA 408 for 2 h,followed by treatment with 20 ng/mL of mouse IFNγ for 24 h. Nitrite (NO₂⁻) levels in the media were measured as a surrogate for nitric oxideusing the Griess Reagent System (cat #G2930, Promega), according to themanufacturer's instructions, since nitrite is a primary, stablebreakdown product of NO. Cell viability was assessed using the WST-1Cell Proliferation Reagent (cat #11644807001, Roche Applied Science)according to the manufacturer's instructions. IC₅₀ values weredetermined based on the suppression of IFNγ-induced nitric oxideproduction normalized to cell viability. Treatment with RTA 408 resultedin a dose-dependent suppression of IFNγ-induced NO production, with anaverage IC₅₀ value of 3.8±1.2 nM. Results from a representativeexperiment are shown in FIG. 1. The IC₅₀ value for RTA 408 was found tobe 45%-65% lower than the IC₅₀ values for compounds 63170 (8±3 nM),63171 (6.9±0.6 nM), 63179 (11±2 nm), and 63189 (7±2 nM). 63170, 63171,63179, and 63189 are compounds of the formulas:

2. Effect of RTA 408 on Nrf2 Target Genes

RTA 408 was tested in two different luciferase reporter assays to assessactivation of the ARE. The first luciferase reporter tested was underthe control of a single ARE derived from the promoter of the human NQO1gene, which allows for quantitative assessment of the endogenousactivity of the Nrf2 transcription factor in cultured mammalian cells.Expression of Firefly luciferase from NQO1-ARE luciferase reporterplasmid is controlled by binding of Nrf2 to a specific enhancer sequencecorresponding to the antioxidant response element (ARE) that wasidentified in the promoter region of the human NADPH:quinoneoxidoreductase 1 (NQO1) gene (Xie et al., 1995). The NQO1-ARE-luciferasereporter plasmid was constructed by inserting the human NQO1-ARE(5′-CAGTCACAGTGACTCAGCAGAATCTG-3′) into the pLuc-MCS vector usingHindIII/XhoI cloning sites (GenScript Corp., Piscataway, N.J.). TheHuH-7 human hepatoma cell line, maintained in DMEM (Invitrogen)supplemented with 10% FBS and 100 U/mL (each) of penicillin andstreptomycin, was transiently transfected using Lipofectamine 2000(Invitrogen) with the NQO1-ARE luciferase reporter plasmid and thepRL-TK plasmid, which constitutively expresses Renilla luciferase and isused as an internal control for normalization of transfection levels.Thirty hours of transfection, cells were treated with RTA 408 for 18 h.Firefly and Renilla luciferase activity was assayed by Dual-GloLuciferase Assay (cat. # E2920, Promega), and the luminescence signalwas measured on an L-Max II luminometer (Molecular Devices). Fireflyluciferase activity was normalized to the Renilla activity, and foldinduction over a vehicle control (DMSO) of normalized Firefly activitywas calculated. FIG. 2 a shows a dose-dependent induction of luciferaseactivity by RTA 408 in this cell line. Values represent the average ofthree independent experiments. Twenty percent less RTA 408 (12 nM) than63189 (14.9 nM) was required to increase transcription from the NQO1 AREin HuH-7 cells by 2-fold. Likewise, 2.1-2.4 fold less RTA 408 than 63170(25.2 nM) and 63179 (29.1 nM), respectively, was required to increasetranscription from the NQO1 ARE in HuH-7 cells by 2-fold.

The effect of RTA 408 on luciferase reporter activation was alsoassessed in the AREc32 reporter cell line. This cell line is derivedfrom human breast carcinoma MCF-7 cells and is stably transfected with aFirefly luciferase reporter gene under the transcriptional control ofeight copies of the rat GSTA2 ARE sequence (Wang, et al., 2006, which isincorporated herein by reference). Following treatment with RTA 408 for18 h, Firefly luciferase activity was measured using the ONE-GloLuciferase Assay System (Promega, Catalog #E6110) according to themanufacturer's instructions. A dose-dependent response was observed inthe AREc32 reporter cell line (FIG. 2 b). A ˜2-fold induction ofluciferase activity was evident following treatment with 15.6 nM RTA 408in both the NQO1-ARE and GSTA2-ARE reporter assay system. When lookingat the results from the GSTA2-ARE (AREc32) luciferase activity study,the effects of 63415 (RTA 408) on GSTA2-ARE induction can be directlycompared to that of RTA 402, 63170, 63171, 63179, and 63189 along withWST1 viability studies (FIGS. 3 a-f). Compared to the values of RTA 402,63415 showed the quickest induction of GSTA2-ARE-mediated transcriptionof the five comparison compounds with a concentration of 93 nM needed toreach 4-fold induction in the luciferase reporter assay. All othercompounds showed a similar induction only with much higherconcentrations with 63170 needing a concentration of 171 nM, 63171needing a concentration of 133 nM, 63179 needing a concentration of 303nM and 63189 needing a concentration of 174 nM to achieve a 4 foldinduction of luciferase activity. These values correspond to a 1.86(63415), 3.40 (63170), 2.65 (63171), 6.05 (63179) and 3.47 (63189) foldincrease in the amount of the active compound needed compared to RTA 402to lead to the same amount of activity.

RTA 408 was also shown to increase transcript levels of known Nrf2target genes in the HFL1 human fetal lung fibroblast and BEAS-2B humanbronchial epithelial cell lines. HFL1 cells were cultured in F-12K mediasupplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.BEAS-2B cells were cultured in DMEM/F-12 media supplemented with 10%fetal bovine serum and 1% penicillin-streptomycin. Cells were plated in6-well dishes at a density of 2.5×10⁵ cells/well. The following day,cells were treated with DMSO (vehicle) or RTA 408 (7.8, 15.6, 31.3,62.5, or 125 nM) for 18 h. Each well received the same amount ofvehicle. Following treatment, media was removed and cells were harvestedusing RLT buffer (Qiagen). Lysates were homogenized using QIAShreddercolumns (Qiagen, Catalog #79654) and RNA was isolated using RNeasy Minikits (Qiagen, Catalog #74104). For reverse transcription, RNA (1 μg) wascombined with Oligo(dT)₁₂₋₁₈ primer and H₂O in a final volume of 23.25μL. The mixture was heated to 70° C. for 10 min and then placed on ice.A master mix containing 8 μL 5×1^(st) strand buffer, 2 μL mg/ml BSA, 2μL 20 mM DTT, 4 μL 5 mM dNTP mix, 0.25 μL RNaseOUT™ and 0.5 μLSuperscript® II reverse transcriptase was added to the RNA mixture andincubated at 42° C. for 1 h. The reaction was inactivated by heating to70° C. for 10 min. The reaction mixture was diluted 1:3 with H₂O priorto use in qPCR. 2.5 μL of the diluted reverse transcription reaction wascombined with one set of PCR primers (0.36 μM final concentration), 2×iQ™ SYBR® Green Supermix (Bio-Rad, Catalog #170-8885) and H₂O to a finalvolume of 20 pt. Sequences for PCR primers are as follows:Glutamate-cysteine ligase, modifier subunit (GCLM), forward primer5′-GCTGTGGCTACTGCGGTATT-3′ (SEQ ID NO: 1) reverse primer5′-ATCTGCCTCAATGACACCAT-3′ (SEQ ID NO: 2); Heme oxygenase-1 (HMOX1)forward primer 5′-TCCGATGGGTCCTTACACTC-3′ (SEQ ID NO: 3), reverse primer5′-TAGGCTCCTTCCTCCTTTCC-3′ (SEQ ID NO: 4); NAD(P)H dehydrogenase,quinone 1 (NQO1) forward primer 5′-AAAACACTGCCCTCTTGTGG-3′ (SEQ ID NO:5), reverse primer 5′-GTGCCAGTCAGCATCTGGTA-3′ (SEQ ID NO: 6); Ribosomalprotein S9 (RPS9) forward primer 5′-GATGAGAAGGACCCCACGGCGTCTG-3′ (SEQ IDNO: 7), reverse primer 5′-GAGACAATCCAGCAGCCCAGGAGGG-3′ (SEQ ID NO: 8);Thioredoxin Reductase 1 (TXNRD1) forward primer5′-ATTGCCACTGGTGAAAGACC-3′ (SEQ ID NO: 9), reverse primer5′-ACCAATTTTGTTGGCCATGT-3′ (SEQ ID NO: 10). All primers had previouslybeen validated for specificity and amplification efficiency. cDNA wasamplified using the following cycle conditions: (95° C. for 3 min, 44cycles of 95° C. for 30 sec, 60° C. for 15 sec, 72° C. for 15 sec,followed by a melt curve of 55° C. to 95° C. in increments of 0.5° C.).The relative abundance of each Nrf2 target gene was determined using thecomparative C_(T) method (ΔΔC_(T)). PCR reactions were run in triplicatewells for each sample. Two independent experiments were performed usingthe conditions described above. Treatment of HFL1 lung fibroblasts withRTA 408 for 18 h resulted in increased expression of several Nrf2 targetgenes, including NQO1, HMOX1, GCLM, and TXNRD1, as measured byquantitative PCR (FIGS. 4 a-d). For all genes tested, induction by RTA408 was dose-dependent and evident at concentrations as low as 15.6 nM.Treatment of BEAS-2B bronchial epithelial cells with RTA 408 for 18 hresulted in a similar dose-dependent increase of all Nrf2 target genesevaluated (FIGS. 5 a-d). RTA 408 also increased expression of Nrf2target genes in normal human mesangial cells (nHMC), the mouse BV2microglial cell line, and the human SH-SY5Y neuroblastoma cell line atsimilar concentrations.

Protein levels of Nrf2 targets NQO1 and HMOX1 were measured in SH-5Y5Yand BV-2 cells by Western blot following treatment with RTA 408. SH-SY5Ycells were plated in 6-well plates at a density of 4×10⁵ cells per well.BV-2 cells were plated in E-well plates at a density of 2.5×10⁴ cellsper well. Twenty-four (BV-2) or 48 (SH-SY5Y) h after plating, cells weretreated with RTA 408 for 24 hours. Following treatment, cells werewashed twice with cold PBS and harvested in lysis buffer. Cells weresonicated and debris was cleared by centrifugation (10 min @ 18,000 rcf,Beckman Coulter, microfuge 18 centrifuge). Total protein in supernatantwas quantified using Bio-Rad protein reagent with BSA as a standard.Equal amounts of total cellular protein were separated on SDS-PAGE, andproteins were transferred to nitrocellulose membrane. Membranes wereblocked for 1 hour in TBST (1×TBS with 0.1% Tween-20) containing 5%milk, washed 3 times with TBST, and incubated with primary antibodiesovernight at 4° C. NQO1 antibody was from Abcam (#AB2346); HMOX1 (HO-1)antibody was from Santa Cruz (#sc-10789); actin antibody was fromMillipore (#MAB 1501). After washing with TBST, secondary antibodieswere added in TBST+5% milk for 1 h at room temperature. AffiniPure goatanti-rabbit or anti-mouse IgG secondary antibodies were from JacksonImmunoResearch (catalog #111-035-144 and #115-035-146, respectively).Membranes were washed in TBST, developed using ECL, and exposed to X-rayfilm. Treatment with RTA 408 also increased NQO1 protein levels inSH-SY5Y cells in a dose-dependent manner (FIG. 6 a). HMOX1 protein wasnot detected in untreated or RTA 408-treated SH-SY5Y cells. In BV2cells, treatment with RTA 408 increased NQO1 and HMOX1 protein levels atconcentrations up to 125 nM (FIG. 6 b). The EC₅₀ value for induction ofNrf2 protein expression in SK-N-SH cells by RTA 408 (56.4 nM) was45%-65% lower than the EC₅₀ values for 63171 (122 nM), 63189 (102 nM),and 63179 (126 nM). The same amount of 63170 (54.6 nM) was required.

The EC₅₀ was measured using an in-cell western NQO1 assay where thecells were incubated with the compound under evaluation for three days.After incubation with the compound of interest, the cells were reactedwith mouse NQO1 antibody and then the next day the cells were reactedwith IRDye-800CW-anti-mouse IgG antibody. The target signals werevisualized and then analyzed.

Consistent with induction of Nrf2 target genes and corresponding proteinproducts, treatment of RAW264.7 mouse macrophage cells for 24 hincreased NQO1 enzymatic activity in a dose-dependent manner, withincreases evident at 7.8 nM (FIG. 7). NQO1 enzymatic activity wasmeasured by a modified Prochaska assay (Prochaska and Santamaria, AnalBiochem. 169:328-336, 1988, which is incorporated herein by reference).

Taken together, these data from multiple cell lines demonstrate thattreatment with RTA 408 increases transcriptional activity controlled byantioxidant response elements, increases expression of Nrf2 targetgenes, and increases the activity of NQO1, an Nrf2 target gene product.

3. Effect of RTA 408 on Markers of Cellular Redox Capacity

Glutathione and NADPH are critical factors required for the maintenanceof cellular redox capacity. Several genes involved in the synthesis ofglutathione (e.g., GCLC and GLCM) and NADPH [e.g., hexose-6-phosphatedehydrogenase (H6PD) and malic enzyme 1 (ME1)] have been demonstrated tobe regulated by Nrf2 (Wu, 2011). The effect of RTA 408 treatment ontotal glutathione levels was evaluated in the mouse AML-12 hepatocytecell line using the GSH-Glo™ Glutathione Assay kit (Promega, Catalog#V6912) according to the manufacturer's instructions. Treatment ofAML-12 cells for 24 h with RTA 408 increased total cellular glutathionelevels in a dose-dependent manner (FIG. 8). Data shown arerepresentative of two independent experiments. A >2-fold increase intotal glutathione was observed at RTA 408 concentrations as low as 15.6nM. The EC₅₀ value using a RAW264.7 mouse model for induction ofglutathione levels by RTA 408 (9.9 nM) was 22%-57% lower than the EC₅₀values for 63170 (12.1 nM), 63171 (23.2 nM), and 63189 (16 nM).

The effect of RTA 408 treatment on the levels of NADPH, as measured bythe absorbance of a redox-sensitive dye, WST-1 (Roche Applied Science,Catalog #11644807001), was evaluated in HCT-116 cells. WST-1 absorbanceis commonly used to assess cell viability by measuring glycolyticproduction of NAD(P)H by viable cells. Therefore, in situations whereNADPH production increases in the absence of any effect on cellviability, WST-1 absorbance also increases (Berridge et al. Biochemica4:14-19, 1996, which is incorporated herein by reference). Several keygenes involved in NADPH production have also been shown to be regulatedby Nrf2 (Thimmulappa et al., 2002; Wu et. al., 2011, which are bothincorporated herein by reference). RTA 408 treatment for 24 h increasedWST-1 absorbance in a dose-dependent manner (FIG. 9), suggesting thatNADPH levels were increased.

The effect of RTA 408 on the expression of genes involved in NADPHsynthesis pathways was also evaluated in this study. HCT-116 cells weretreated with RTA 408 for 24 h, and mRNA levels of H6PD, phosphogluconatedehydrogenase (PGD), transketolase (TKT), and ME1 were measured usingquantitative PCR. HCT-116 cells were plated in 6-well dishes at adensity of 3×10⁵ cells/well. The following day, cells were treated withDMSO (vehicle), 10 nM RTA 408, or 50 nM RTA 408 for 24 h. Each wellreceived the same amount of vehicle. Following treatment, media wasremoved and cells were harvested using RLT buffer (Qiagen). Lysates werehomogenized using QIAShredder columns (Qiagen, Catalog #79654) and RNAwas isolated using RNeasy Mini kits (Qiagen, Catalog #74104). Forreverse transcription, RNA (1 μg) was combined with Oligo(dT)12-18primer and H₂O in a final volume of 23.25 μL. The mixture was heated to70° C. for 10 min and then placed on ice. A master mix containing 8 μL5×1st strand buffer, 2 μL 1 mg/ml BSA, 2 μL 20 mM DTT, 4 μL 5 mM dNTPmix, 0.25 μL RNaseOUT™ and 0.5 μL Superscript® II reverse transcriptasewas added to the RNA mixture and incubated at 42° C. for 1 h. Thereaction was inactivated by heating to 70° C. for 10 min. The reactionmixture was diluted 1:3 with H₂O prior to use in qPCR. 2.5 μL of thediluted reverse transcription reaction was combined with one set of PCRprimers (0.36 μM final concentration), 2× iQ™ SYBR® Green Supermix(Bio-Rad, Catalog #170-8885) and H₂O to a final volume of 20 μL.Sequences for PCR primers are as follows: Ribosomal protein S9 (RPS9)forward primer 5′-GATGAGAAGGACCCCACGGCGTCTG-3′ (SEQ ID NO: 7), reverseprimer 5′-GAGACAATCCAGCAGCCCAGGAGGG-3′ (SEQ ID NO: 8);Hexose-6-phosphate dehydrogenase (H6PD) forward primer5′-GAGGCCGTGTACACCAAGAT-3′ (SEQ ID NO: 11), reverse primer5′-AGCAGTGGGGTGAAAATACG-3′ (SEQ ID NO: 12), Phosphogluconatedehydrogenase (PGD) forward primer 5′-AAGGCACTCTACGCTTCCAA-3′ (SEQ IDNO: 13), reverse primer 5′-AGGAGTCCTGGCAGTTTTCA-3′ (SEQ ID NO: 14),Transketolase (TKT) forward primer 5′-CATCTCCGAGAGCAACATCA-3′ (SEQ IDNO: 15), reverse primer 5′-TTGTATTGGCGGCTAGTTCC-3′ (SEQ ID NO: 16);Malic enzyme 1 (ME1) forward primer 5′-TATATCCTGGCCAAGGCAAC-3′ (SEQ IDNO: 17) reverse primer 5′-GGATAAAGCCGACCCTCTTC-3′ (SEQ ID NO: 18). Allprimers had previously been validated for specificity and amplificationefficiency. cDNA was amplified using the following cycle conditions:(95° C. for 3 min, 44 cycles of 95° C. for 30 sec, 60° C. for 15 sec,72° C. for 15 sec, followed by a melt curve of 55° C. to 95° C. inincrements of 0.5° C.). The relative abundance of each target gene wasdetermined using the comparative CT method (ΔΔCT). PCR reactions wererun in triplicate wells for each sample. Two independent experimentswere performed using the conditions described above. Treatment with RTA408 resulted in a dose-dependent increase in expression of genesinvolved in NADPH synthesis (FIGS. 10 a-d).

In summary, treatment with RTA 408 increased total glutathione levels inAML-12 hepatocytes and increased WST-1 absorbance, a marker of NADPHproduction, in HCT-116 cells. This observation correlated with anincrease in the expression of several key genes encoding enzymesinvolved in NADPH synthesis.

4. Effect of RTA 408 on TNFα-induced NF-κB Signaling

NF-κB is a transcription factor that plays a central role in theregulation of many immune and inflammatory responses. RTA 402 and otherAIMs have been shown to inhibit pro-inflammatory NF-κB signaling in avariety of cell lines (Shishodia, 2006; Ahmad, 2006; Yore, 2006). Usingthe mouse NIH3T3/NF-κB-luc cell line (Panomics), the effects of RTA 408and the compounds 63171, 63179, 63170, and 63189 on the NF-κB-Lucreporter were explored. The NIH3T3/NF-κB-luc cell line maintains achromosomal integration of a Firefly luciferase reporter constructregulated by eight copies of the NF-κB response element. The effects ofthese compounds can be quantified by measuring the value of the NF-κBIC₅₀. RTA 408 showed a 1.2 μM IC₅₀, which when normalized for viabilityshowed an IC₅₀ of 1.4 μM. The other four compounds showed NIH3T3/NF-κBIC₅₀ values of 1.7, 0.2, 1.1, and 1.1 μM, which when viabilitynormalized showed IC₅₀ values of 1.8, 0.6, 1.1, and 1.0 μM,respectively. RTA 408 and its effects on NF-κB are plotted as a functionof dosing and relative fold change as well as WST1 and WST1/2 are shownin FIGS. 11 a & b. The effect of RTA 408 on TNF-α-induced NF-κBsignaling was evaluated in HeLa/NF-κB-Luc cells, a human cervicaladenocarcinoma cell line stably transfected with a luciferase reporterconstruct under the control of multiple NF-κB transcriptional responseelements. HeLa/NF-κB-Luc cells were pretreated for 1 h with RTA 408,followed by treatment with TNF-α (10 ng/mL) for an additional 5 h. Aftertreatment, luminescence was measured, and the effect of RTA 408pretreatment on TNF-α-induced luciferase activity was determined. Theaverage results and standard deviations from three independentexperiments are shown in FIG. 12. RTA 408 dose-dependently inhibitedTNFα-induced NF-κB activation with an IC₅₀ value of 517±83 nM. Similarresults were observed in another NF-κB reporter cell line(A549/NF-κB-Luc) where RTA 408 inhibited TNFα-induced NF-κB activationwith an IC₅₀ value of 627 nM (range 614-649 nM). RTA 408 was 1.6-1.8fold more efficient at reducing expression from the NF-κB promoterreporter in HeLa/NF-κB-Luc cells than 63189 (854 nM) and 63170 (953 nM),respectively. Further experimentation with the human A549 cell lineshowed an IC₅₀ for RTA 408 as 1.7 μM and a value that has been viabilitynormalized to 1.7 μM. The IC₅₀ of RTA 408 showed similar activity to63189, 63179, 63171, and 63170 which showed IC₅₀ values of 1.1, 1.4,2.0, and 1.0, respectively. When those values were viability normalized,the assay showed 1.2, 1.5, 2.1 and 1.1 μM IC₅₀, respectively. The foldchange for NF-κB as a function of RTA 408 concentration along with WST1and WST1/2 curves were plotted and are shown in FIGS. 13 a & b.

The effect of RTA 408 on TNFα-induced phosphorylation of IκBα, a keystep in activation of the NF-κB pathway, was also evaluated in HeLacells. HeLa cells were pretreated with RTA 408 for 6 h, followed bytreatment with TNF-α (20 ng/mL) for 5 min. Total and phosphorylatedlevels of IκBα were evaluated by Western blot. Primary IκBα antibodieswere from Santa-Cruz (sc-371), pIκBα antibody was from Cell Signaling(9246), actin antibody was from Millipore (MAB 1501).Peroxidase-conjugated affini-pure Goat anti-Rabbit (IgG) andperoxidase-conjugated affini-pure Goat anti-Mouse IgG secondaryantibodies were purchased from Jackson ImmunoResearch. Protein blotswere developed using ECL, and exposed to X-ray film. Consistent with theresults from the luciferase reporter assay, RTA 408 inhibitedTNF-α-induced phosphorylation of IκBα in a dose-dependent manner (FIG.14).

RTA 408 has also been demonstrated to inhibit other pro-inflammatorysignaling pathways, such as IL-6-induced signal transducer and activatorof transcription 3 (STAT3) phosphorylation and receptor activator ofNF-κB ligand (RANKL)-induced osteoclastogenesis. In HeLa cells,pretreatment with 1 μM RTA 408 for 6 h inhibited phosphorylation ofSTAT3 induced by IL-6. STAT3 (124H6) and phospho-STAT3 (Tyr705)monoclonal antibodies were from Cell Signaling Technology.Peroxidase-conjugated Affini-pure Goat anti-Rabbit IgG andPeroxidase-conjugated Affini-pure Goat anti-Mouse IgG were from JacksonImmunoResearch. Osteoclastogenesis is a multi-step differentiationprocess that results from the binding of RANKL to its receptor, RANK, oncells of hematopoietic origin. This results in the activation of NF-κBand MAPK, which in turn increase transcription of osteoclast-specifictarget genes, including tartrate-resistant acid phosphatase (TRAP). Theeffect of RTA 408 on RANKL-induced osteoclastogenesis was evaluated inthe mouse macrophage cell line RAW264.7. RAW 264.7 cells were plated in24-well plates at a density of 5,000 cells/well. The next day, cellswere treated with RTA 408 for 2 h and then treated with 50 ng/mLrecombinant mouse RANKL (R&D systems). The treated cells were incubatedfor four days to allow differentiation into osteoclasts. Differentiationinto osteoclasts was assessed by measuring TRAP activity. In brief, 90μl of conditioned cell culture media was removed from each test well andaliquoted into triplicate wells (30 μL/well) of a 96-well plate. 170 μLof TRAP Assay buffer (Kamiya Biomedical) was then added to each well andthe plate was incubated at 37° C. for 3 hours. Following the incubation,absorbance at 540 nm was determined using a Spectramax M2 plate readingspectrophotometer. RTA 408 dose-dependently inhibited RANKL-induced TRAPactivity and the formation of osteoclasts, with an IC₅₀ of ˜5-10 nM.

5. Effect of RTA 408 on Expression of Genes Encoding TransaminaseEnzymes

Transaminase elevations were observed in the 28-day toxicity studieswith RTA 408 in rats and, to a much lower extent, in monkeys. Similarfindings have been observed following oral administration of a relatedAIM (bardoxolone methyl) in humans (Pergola, 2011). One hypothesis forthis effect is that AIMs directly or indirectly increase transaminasegene expression in the absence of cellular toxicity. To assess whethertreatment with RTA 408 affects transaminase mRNA levels, mouse AML-12hepatocytes were treated with RTA 408 for 18 h, and the mRNA levels ofgenes encoding transaminases were measured using quantitative PCR.AML-12 cells were plated in E-well culture dishes at 3×10⁵ cells perwell using 2 mL of media per well. The following day cells were treatedwith DMSO (vehicle) or 250 nM and 500 nM RTA 408 for 18 h at 37° C. Eachwell received 0.1% DMSO. Three independent replicate experiments wereperformed. Following treatment, media was removed and cells wereharvested using RLT buffer (Qiagen). Lysates were homogenized usingQIAShredder columns (Qiagen, Catalog #79654) and RNA was isolated usingRNeasy Mini kits (Qiagen, Catalog #74104). For reverse transcription,RNA (1 μg) was combined with Oligo(dT)12-18 primer and H₂O in a finalvolume of 23.25 μL. The mixture was heated to 70° C. for 10 min and thenplaced on ice. A master mix containing 8 μL 5×1st strand buffer, 2 μL 1mg/ml BSA, 2 μL 20 mM DTT, 4 μL 5 mM dNTP mix, 0.25 μL RNaseOUT™ and 0.5μL Superscript® II reverse transcriptase was added to the RNA mixtureand incubated at 42° C. for 1 h. The reaction was inactivated by heatingto 70° C. for 10 min. The reaction mixture was diluted 1:3 with H₂Oprior to use in qPCR. 2.5 μL of the diluted reverse transcriptionreaction was combined with one set of PCR primers (0.36 μM finalconcentration), 2× iQ™ SYBR® Green Supermix (Bio-Rad, Catalog #170-8885)and H₂O to a final volume of 20 μL. Sequences for PCR primers are asfollows: Ribosomal protein L19 (Rpl19) forward primer5′-TCAGGCTACAGAAGAGGCTTGC-3′ (SEQ ID NO: 19), reverse primer5′-ACAGTCACAGGCTTGCGGATG-3′ (SEQ ID NO: 20); NAD(P)H dehydrogenase,quinone 1 (Nqo1) forward primer 5′-TCGGGCTAGTCCCAGTTAGA-3′ (SEQ ID NO:21), reverse primer 5′-AAAGAGCTGGAGAGCCAACC-3′ (SEQ ID NO: 22); Glutamicpyruvic transaminase 1 (Gpt1 or Alt1) forward primer5′-CACGGAGCAGGTCTTCAACG-3′ (SEQ ID NO: 23), reverse primer5′-AGAATGGTCATCCGGAAATG-3′ (SEQ ID NO: 24); Glutamic pyruvictransaminase 2 (Gpt2 or Alt2) forward primer 5′-CGCGGTGCAGGTCAACTACT-3′(SEQ ID NO: 25), reverse primer 5′-CCTCATCAGCCAGGAGAAAA-3′ (SEQ ID NO:26); Glutamate oxaloacetate transaminase 1 (Got1 or Ast1) forward primer5′-GGCTATTCGCTATTTTGTGT-3′ (SEQ ID NO: 27), reverse primer5′-GACCAGGTGATTCGTACAAT-3′ (SEQ ID NO: 28); Glutamate oxaloacetatetransaminase 2 (Got2 or Ast2) forward primer 5′-AGAGTCCTCTTCAGTCATTG-3′(SEQ ID NO: 29), reverse primer 5′-ATGATTAGAGCAGATGGTGG-3′ (SEQ ID NO:30). All primers had previously been validated for specificity andamplification efficiency. cDNA was amplified using the following cycleconditions: (95° C. for 3 min, 44 cycles of 95° C. for 30 sec, 60° C.for 15 sec, 72° C. for 15 sec, followed by a melt curve of 55° C. to 95°C. in increments of 0.5° C.). The relative abundance of each target genewas determined using the comparative CT method (ΔΔCT). PCR reactionswere run in triplicate wells for each sample. Treatment with RTA 408increased mRNA levels of alanine transaminase 1 (Alt1 or Gpt1) andaspartate transaminase 1 (Ast1 or Got1) (FIGS. 15 a,c). RTA 408 had noeffect on alanine transaminase 2 (Alt2 or Gpt2) mRNA levels and reducedmRNA levels of aspartate transaminase 2 (Ast2 or Got2) (FIGS. 15 b,d).These results demonstrate that RTA 408, at the concentrations tested(250 nM or 500 nM), affects transaminase gene expression in vitro.

6. Effect of RTA 408 on Levels of Glycolytic Intermediates

Studies in diabetic mice have demonstrated that bardoxolone methylincreases muscle-specific insulin-stimulated glucose uptake (Saha,2010). In humans, a higher percentage of patients receiving bardoxolonemethyl reported experiencing muscle cramps compared with patientsreceiving placebo (Pergola, 2011). Muscle spasms have also been reportedin diabetic patients following insulin administration, suggesting apossible association with muscle glucose metabolism. The effect of RTA408 on glycolytic metabolism was evaluated through the assessment oflactate and pyruvate levels in cultured rodent C2C12 muscle cells. Tomeasure lactate levels, differentiated C2C12 myotubes were treated with1 μM or 2 μM RTA 408 or insulin for 3 h at 37° C. Buffer was removed andsaved for measurement of extracellular lactate levels. Cell debris waspelleted by centrifugation (10 min at 14,000 rpm) prior to measurementof lactate. To measure intracellular lactate, cells were suspended in0.1% Triton X-100 in PBS and lysed by shearing with a 25 gauge needle.Cell lysate was centrifuged (10 min at 14,000 rpm, 4° C.) and lactatewas measured in the supernatant. Intracellular and extracellular lactatewas measured using the Lactate Assay Kit (BioVision, Catalog #K607-100).Similar to treatment with insulin, treatment of differentiated C2C12myotubes with 1 μM or 2 μM RTA 408 for 3 h significantly increasedintracellular and extracellular lactate levels in a dose-dependentmanner.

To measure pyruvate levels, differentiated C2C12 myotubes were treatedwith 250 or 500 nM RTA 408 or 100 nM insulin for 18 h. Following drugtreatment, media was removed and cells were washed with PBS. Cells werelysed in Pyruvate Assay Buffer (Pyruvate Assay Kit, BioVision, Catalog#K609-100). Cell lysates were centrifuged (10 min at 14,000 rpm, 4° C.)and pyruvate levels were measured in the supernatant. Treatment of C2C12differentiated myotubes with 250 nM or 500 nM RTA 408 for 18 h alsosignificantly (P<0.0001, noted by asterisks) increased intracellularpyruvate levels in a dose-dependent manner (FIG. 16). Together, theseresults demonstrate that RTA 408, at the concentrations tested, canaffect muscle glycolytic intermediates in vitro; however, it is unclearhow the results from this in vitro system at the RTA 408 concentrationstested relate to the potential effects on glucose metabolism atclinically-relevant dose levels in humans.

7. In Vitro Evaluation of RTA 408 Efflux by MRP-1

One of characteristic of a drug candidate is the compound's effluxratio. The efflux ratio measures how easily the compound is transportedacross a membrane. The MRP-1 protein, or the multidrugresistance-assistance protein 1, is one of a family of proteins whichhelp to facilitate the transport of organic anions and other smallmolecules through cellular membranes. A larger efflux ratio typicallymeans that the drug candidate is more readily transported out of themembrane and less available to modulate intracellular processes. Similarproteins also regulate the transport of compounds across the blood-brainbarrier. The efflux ratio MRP-1 for RTA 408 (1.3) was experimentallydetermined to be approximately ten-fold lower than 63170 (10) and 63171(11.2) and over 40-fold lower than 63179 (56.5) and 63189 (57.1).Without being bound by theory, RTA 408 may not be a good substrate forMRP-1 and/or a candidate for p-glycoprotein mediated efflux at theblood-brain barrier. In some embodiments, RTA 408 may be used fortreating disorders of the central nervous system (CNS).

C. Protective Effects of RTA 408 in Animal Models of Lung Disease

RTA 408 was tested in several animal models of pulmonary disease toevaluate its potential efficacy in the lung. For all studies, RTA 408was orally administered daily in sesame oil at dose levels in the rangeof 3 to 150 mg/kg. In most cases, RTA 408 was administered startingseveral days prior to the induction of the lung injury response.

1. LPS-induced Pulmonary Inflammation in Mice

RTA 408 was tested in two studies of LPS-induced pulmonary inflammationin mice. In the first study, intended to be a preliminary dose-rangefinder, RTA 408 (30, 100, or 150 mg/kg) was administered orally oncedaily for three days, followed by LPS administration 1 h after the finaldose. Bronchoalveolar lavage fluid (BALF) was collected 20 h after LPSadministration (21 h after the final dose of RTA 408) and evaluated forlevels of pro-inflammatory markers (i.e., IL-6, IL-12p40, TNF-α, andRANTES) using Luminex™ technology. RTA 408 treatment resulted in asignificant reduction in IL-12p40 at all doses and in TNF-α at the 100and 150 mg/kg doses (FIG. 17). In the second study, RTA 408 (10, 30, or100 mg/kg) was administered daily for six days, followed by LPSadministration 1 h after the final dose. In this study, significantdecreases in body weight were observed at the 100 mg/kg dose levelstarting on Day 3. Significant reductions in TNF-α were observed at the10 mg/kg dose, and significant reductions in IL-12p40, TNF-α, and RANTESwere observed at the 30 mg/kg dose (FIG. 18 a). Further evaluation oflungs from mice in this study revealed meaningful engagement of relevantNrf2 target genes, including significant induction of NQO1 enzymeactivity (by measurement of rate of reduction of2,6-dicholorphenol-indophenol) and increases in total GSH (GSH-Glo™,Promega, Madison, Wis.) at 10 and 30 mg/kg (FIG. 18 b).

2. Bleomycin-Induced Pulmonary Fibrosis

The effect of RTA 408 was also evaluated in models of bleomycin-inducedpulmonary fibrosis in mice and rats. In the first preliminary study, RTA408 (10, 30, or 100 mg/kg) was administered to mice daily via oralgavage for 39 days, with bleomycin challenge (intranasal) on day 10. Onthe last day of dosing, lung tissue was collected and histology wasperformed to evaluate the extent of inflammation and interstitialfibrosis. In this model, no statistically significant effects wereobserved at the RTA 408 doses tested (FIGS. 19 a & b). Additionalevaluation was performed using a rat model of pulmonary fibrosis thathas been extensively characterized at the Lovelace Respiratory ResearchInstitute. In this study, rats were challenged with bleomycin or salineby intratracheal administration on day 0. Following the challenge,animals received RTA 408 (3, 10, or 30 mg/kg) daily via oral gavage for28 days. Administration of the 30-mg/kg dose was stopped on day 14 dueto excessive dehydration and diarrhea in the animals. For the remaininganimals, bronchoalveolar lavage fluid was collected on day 28 forassessment of pro-inflammatory infiltrates by flow cytometry, and lungtissue was analyzed for hydroxyproline levels by LC-MS andhistopathology. Challenge with bleomycin sulfate induced a substantialrelease of neutrophils and an increase in soluble collagen in the BALF,as well as an increase in hydroxyproline in the lung. Treatment with 3and 10 mg/kg RTA 408 significantly suppressed polymorphonuclear (PMN)cell infiltration into the lungs and also produced a meaningfulreduction (˜10%-20%) in hydroxyproline deposition (FIGS. 20 a & b).

Importantly, histopathological evaluation revealed a significantdecrease in collagen deposition, as assessed by trichrome staining, inrats treated with RTA 408. Whereas bleomycin control animals primarilyexhibited moderate staining, animals treated with 10 mg/kg RTA 408 hadpredominantly minimal to mild staining (Table 2).

TABLE 2 Effect of RTA 408 on collagen deposition in rat lung as assessedby intensity of trichrome staining RTA 408 RTA 408 StainingIntensity^(a) Bleomycin Control (3 mg/kg) (10 mg/kg) Minimal 0 0 3 Mild1 0 4 Moderate 7 7 1 ^(a)Values represent intensity of staining inanimals with interstitial trichrome staining in areas ofbleomycin-induced lung alterations.

Further evaluation of lungs from rats in this study also revealedmeaningful engagement of relevant Nrf2 target genes as assayed byQuantigene Plex 2.0 Multiplex assay (Affymetrix, Santa Clara, Calif.)(FIG. 21). RTA 408 significantly and dose-dependently increased NQO1,Txnrd, Gsr, and Gst enzyme activity in the lungs of rats exposed tobleomycin, demonstrating Nrf2 activation by RTA 408 in this diseasesetting. NQO1 enzyme activity was assessed by measuring the rate ofreduction of DCPIP. Txnrd, Gst, and Gst enzyme activities were measuredusing commercially available kits from Cayman Chemical (Ann Arbor,Mich.).

3. Cigarette Smoke-Induced COPD in Mice

RTA 408 was also tested in a mouse model of cigarette smoke-inducedCOPD. Mice received RTA 408 (3, 10, or 30 mg/kg) daily via oral gavagefor two weeks and were exposed to cigarette smoke five days per weekduring the RTA 408 dosing period. At the end of the study, lung tissueand BALF were collected for analysis of inflammatory infiltrates andcytokines. In this experiment, multiple-dose administration of RTA 408at doses as low as 3 mg/kg RTA 408 resulted in significant suppressionof pro-inflammatory cytokines, including KC (functional mouse homolog ofhuman IL-8) and TNF-α as measured using Luminex™ Technology. A summaryof results from this study is presented in FIGS. 22 a-e. An AIM analog(63355) was tested in the same study for comparison. 63355 is a compoundof the formula:

Further evaluation of lungs from mice in this study also revealedmeaningful engagement of relevant Nrf2 target genes (FIG. 23). NQO1enzyme activity in the lung, measured as the rate of reduction of DCPIP,was significantly decreased by cigarette smoke exposure; administrationof RTA 408 rescued this loss. Txnrd enzyme activity was also induced bythe 30 mg/kg dose of RTA 408. In general Gsr enzyme activity was notaltered, and Gst enzyme activity was decreased with treatment—both ofwhich were likely the consequence of a temporal response for theseenzymes. Txnrd, Gst, and Gst enzyme activities were measured usingcommercially available kits from Cayman Chemical (Ann Arbor, Mich.).

4. Ovalbumin-Induced Asthma in Mice

The potential activity of RTA 408 was also evaluated in a pilot study ina mouse model of ovalbumin-induced asthma. Mice were sensitized with anIP injection of ovalbumin and aluminum hydroxide on Day 0 and Day 14 andchallenged intranasally with ovalbumin in saline on Days 14, 25, 26, and27. Mice received RTA 408 (3, 10, or mg/kg) daily via oral gavage onDays 1-13 and 15-27. Following sensitization and challenge withovalbumin, vehicle-treated mice had a significant increase in the totalnumber of leukocytes compared with positive control(dexamethasone)-treated mice. An increase in the number of T cells and Bcells was also observed in the vehicle-treated mice. Treatment with RTA408 at 30 mg/kg significantly reduced the number and percentage of Bcells within the airways. RTA 408 (3 and 30 mg/kg) also significantlyreduced the number of macrophages, but not the mean percentage ofmacrophages, detected in the airways. These observations are suggestiveof potential efficacy in this model.

5. Effects of RTA 408 on LPS-Induced Sepsis in Mice

Sepsis was induced on Day 0 with an IP injection of LPS (21 mg/kg), andsurvival was followed until Day 4. RTA 408 (10, 30, or 100 mg/kg) wasadministered daily via oral gavage from Day −2 to Day 2. In the vehiclecontrol group, 60% of the animals survived until Day 4 (higher than the˜40% survival rate expected in this model). In the RTA 408 treatmentgroups, 80% of the animals in the 10 mg/kg dose group and 90% of theanimals in the 30 mg/kg dose group survived until Day 4 (FIGS. 24 c &d). For the 100 mg/kg dose group, 90% of the animals survived until Day4, with only a single death occurring on Day 4. Although these RTA408-induced effects are indicative of profound efficacy in this model,the relatively high survival rate in the vehicle control group precludeda statistically-significant difference between the control and RTA408-treated groups. Results obtained using the compound RTA 405 are alsopresented (FIGS. 24 a & b). RTA 405 is a compound of the formula:

6. Effects of RTA 408 Against Radiation-Induced Oral Mucositis

Exposure to acute radiation directed to the buccal cheek pouch ofhamsters produces effects similar to those observed in oral ulcerativemucositis in humans. These effects include moderate to severe mucositischaracterized by severe erythema and vasodilation, erosion of thesuperficial mucosa, and formation of ulcers. A single study wasconducted to evaluate the effects of RTA 408 in this model. On Day 0,each hamster was given an acute radiation dose of 40 Gy directed to theleft buccal cheek pouch. RTA 408 (10, 30, or 100 mg/kg) was orallyadministered twice daily from Day −5 to Day −1, and Day 1 to Day 15.Beginning on Day 6 and continuing until Day 28 on alternate days, oralmucositis was evaluated using a standard 6-point scoring scale. Both the30 and 100 mg/kg doses of RTA 408 caused a significant reduction in theduration of ulcerative mucositis (FIG. 25). Furthermore, adose-dependent decrease in the percentage of animals with mucositisscores ≧3 was also observed. However, administration of RTA 408 at 30 or100 mg/kg caused significant dose-dependent reductions in weight gain inirradiated hamsters. Due to weight loss in excess of 20%, two out ofeight hamsters in the 100 mg/kg dose group were euthanized on Day 2.

7. Effect of RTA 408 on the Induction of Nrf2 Biomarkers In Vivo

As described above, a key molecular target of RTA 408 is Nrf2, a centraltranscriptional regulator of antioxidative cellular protection.Activation of Nrf2 induces upregulation of a battery of cytoprotectivegenes, including NQO1, enzymes involved in GSH synthesis [i.e.,glutamate-cysteine ligase catalytic and modifier subunits (Gclc andGclm)], enzymes involved in detoxification (i.e., glutathioneS-transferases [Gsts]), and efflux transporters [i.e., multidrugresistance-associated proteins (Mrps)]. Induction of these genes resultsin a coordinated cellular effort to protect against oxidative insult,highlighted by increased antioxidative capacity, induction ofglutathione synthesis, and conjugation and export of potentially harmfulmolecules from the cell. In addition to the efficacy endpoints and Nrf2target gene expression evaluated in the various animal models describedabove, the ability of RTA 408 to induce expression of Nrf2 target geneswas also assessed using tissues collected from healthy RTA 408-treatedmice, rats, and monkeys.

As part of the non-GLP 14-day toxicity studies of RTA 408 in mice, rats,and monkeys, tissues were collected for the purposes of measuring mRNAand enzyme activity levels of selected Nrf2 target genes. For mice andrats, liver samples were collected 4 h after the final dose on Day 14.For monkeys, blood (for PBMC isolation), liver, lung, and brain tissuewere collected 24 h after the final dose on Day 14. Enzyme activity forNQO1, Gst, and glutathione reductase (Gsr), as described above, weremeasured in tissue homogenates. Levels of mRNA were determined usingQuantigene Plex 2.0 technology according to the manufacturer's protocol,which involves a hybridization-based assay using xMAP® Luminex® magneticbeads for direct quantification of mRNA targets. In addition, RTA 408concentrations were measured in plasma and tissues by LC/MS/MS methodson a TQD mass spectrometer (Waters, Milford, Mass.).

RTA 408 generally increased the expression of various Nrf2 target genesin a dose-dependent manner at doses of 10, 30, and 100 mg/kg (FIG. 26,FIG. 27 a, FIGS. 28 a & b). Transcriptional upregulation of Nrf2 targetgenes by RTA 408 also resulted in functional increases in theantioxidant response, as manifested by dose-dependent increases in NQO1,Gst, and Gsr enzyme activity in rodent liver, as well as monkey liverand lung (FIGS. 29 a & b, FIGS. 30 a & b, FIGS. 31 a & b). Furthermore,in rodents liver exposure of RTA 408 correlated with the level of enzymeactivity of NQO1, the prototypical target gene for Nrf2 (FIG. 32 b, FIG.33 b). In monkeys, the level of mRNA expression in PBMCs of both NQO1and sulfuredoxin 1 (SRXN1) correlated with plasma exposure to RTA 408(FIGS. 37 a & b). Overall, RTA 408 increased mRNA levels and activity ofNrf2 targets, and such increases generally correlated with tissue andplasma exposures, suggesting Nrf2 targets may serve as feasiblebiomarkers for Nrf2 activation (FIGS. 34 a & b) and may be useful forassessing pharmacological activity of RTA 408 in healthy human subjects.

D. Safety Pharmacology

A GLP-compliant safety pharmacology program was completed using RTA 408.This included in vitro and in vivo (monkey) studies on thecardiovascular system, as well as studies on the respiratory system andcentral nervous system in rats.

1. Evaluation of the Effects of RTA 408 on Cloned hERG ChannelsExpressed in HEK293 Cells

This study was conducted to assess the effects of RTA 408 on the rapidlyactivating inward rectifying potassium current (I_(Kr)) conducted byhERG (human ether-a-go-go-related gene) channels stably expressed in thehuman embryonic kidney (HEK293) cell line. The effects of RTA 408 on thehERG-related potassium current were assessed using whole-cell patchclamp electrophysiology methods. RTA 408 was determined to have IC₅₀value of 12.4 μM in a hERG QPatch_Kv11.1 assay. This value was 2.5-3fold higher than the values for 63170 (4.9 μM) and 63189 (3.8 μM),respectively. The RTA 408 IC₅₀ value was similar to the 63171 value(15.7 μM).

2. Cardiovascular Evaluation of RTA 408 in the Cynomolgus Monkey

A single study was conducted to evaluate the potential cardiovasculareffects of RTA 408 in conscious freely moving cynomolgus monkeys. Thesame four male and four female cynomolgus monkeys were administered thevehicle (sesame oil) and RTA 408 at dose levels of 10, 30, and 100 mg/kgaccording to a Latin square design, with one animal/sex/treatment dosedeach week followed by a 14-day washout period between administrations,until each animal received all treatments. Vehicle and RTA 408 wereadministered to all animals via oral gavage at a dose volume of 5 mL/kg.

Animals were instrumented with telemetry transmitters for measurement ofbody temperature, blood pressure, heart rate, and electrocardiogram(ECG) evaluation. Body temperature, systolic, diastolic, and meanarterial blood pressure, heart rate, and ECG parameters (QRS durationand RR, PR, and QT intervals) were monitored continuously from at least2 h pre-dose until at least 24 h post-dose. ECG tracings were printed atdesignated time points from the cardiovascular monitoring data and werequalitatively evaluated by a board-certified veterinary cardiologist.Prior to the first administration on study, untreated animals werecontinuously monitored for cardiovascular endpoints for at least 24 h,and these data were used in the calculation of the corrected QT intervalthroughout the study.

Observations for morbidity, mortality, injury, and availability of foodand water were conducted at least twice daily for all animals. Clinicalobservations were conducted pre-dose, approximately 4 h post-dose, andfollowing completion of the cardiovascular monitoring period. Bodyweights were measured and recorded on the day prior to each treatmentadministration.

RTA 408 at dose levels of 10, 30, and 100 mg/kg did not producemortality, adverse clinical signs, or result in meaningful changes inbody weight, body temperature, blood pressure, or qualitative orquantitative (PR, RR, QRS, QT intervals) ECG parameters (FIG. 35; Table45). In the 100 mg/kg dose group, a small (1.6% on average) butstatistically significant increase in the corrected QT interval wasobserved; however, individual animal data did not show consistentincreases in QTc that would indicate a test article related effect.Consequently, due to the small magnitude of change and lack of aconsistent response in individual animals, these slight increases in QTcwere not considered to be related to RTA 408 treatment. Therefore, oraladministration of RTA 408 produced no effects on cardiovascular functionin cynomolgus monkeys at doses up to and including 100 mg/kg.

3. Neurobehavioral Evaluation of RTA 408 in Rats

The potential acute neurobehavioral toxicity of RTA 408 was evaluated inrats. Three treatment groups of 10 male and 10 female CD® [Crl:CD® (SD)]rats received RTA 408 at dose levels of 3, 10, or 30 mg/kg. Oneadditional group of 10 animals/sex served as the control and receivedvehicle (sesame oil). Vehicle or RTA 408 was administered to all groupsvia oral gavage once on Day 1 at a dose volume of 10 mL/kg.

Observations for morbidity, mortality, injury, and availability of foodand water were conducted twice daily for all animals. Observations forclinical signs were conducted prior to dosing on Day 1 and followingeach functional observational battery (FOB) evaluation. FOB evaluationswere conducted pre-dose (Day −1) and at approximately 4 and 24 hpost-dose. Body weights were measured and recorded pre-dose on Day 1.

RTA 408 at doses of 3, 10, and 30 mg/kg did not produce mortality,adverse clinical observations, or effects on any of the neurobehavioralmeasures tested. Slight decreases in body weight gain were observedapproximately 24 h after dosing in the 30 mg/kg group that maypotentially be test article-related. With respect to the basicneurobehavioral endpoints evaluated in this study, RTA 408 did notproduce any adverse effects in rats at doses up to and including 30mg/kg.

4. Pulmonary Evaluation of RTA 408 in Rats

The potential effect of RTA 408 on pulmonary function was evaluated inrats. Three treatment groups of eight male and eight female CD® [Crl:CD®(SD)] rats received RTA 408 at dose levels of 3, 10, or 30 mg/kg. Oneadditional group of 8 animals/sex served as the control and receivedvehicle (sesame oil). Vehicle or RTA 408 was administered to all groupsvia oral gavage once on Day 1 at a dose volume of 10 mL/kg.

Observations for mortality, morbidity, injury, and availability of foodand water were conducted twice daily for all animals. Clinicalobservations were conducted prior to dosing, approximately 4 hpost-dose, and following completion of the 8-h pulmonary monitoringperiod. Body weights were measured and recorded on the day of RTA 408administration. Pulmonary function (respiratory rate, tidal volume, andminute volume) was monitored for at least 1 h prior to dosing toestablish a baseline and for at least 8 h post-dose.

RTA 408 at doses of 3, 10, and 30 mg/kg did not produce mortality,adverse clinical observations, or effects on any of the pulmonaryparameters evaluated. Therefore, with respect to the basic pulmonaryendpoints evaluated in this study, RTA 408 did not produce any adverseeffects in rats at doses up to and including 30 mg/kg.

E. Nonclinical Overview

1. Pharmacokinetics

RTA 408 has been investigated both in vitro and in vivo to assess its PKand metabolism properties. In vitro studies have been conducted todetermine RTA 408 plasma protein binding and blood/plasma partitioning,cytochrome P450 (CYP450) inhibition and induction, and to identifymetabolites formed by liver microsomes of mice, rats, monkeys, andhumans. Data pertaining to the in vivo absorption and distributionfollowing repeated administration of RTA 408 has been obtained primarilythrough monitoring of drug levels in plasma and select tissues fromtoxicology studies. Sensitive and selective liquid chromatography-massspectrometry-based bioanalytical methods (LC/MS/MS) have been used tomeasure concentrations of RTA 408 in plasma, blood, and tissues withappropriate accuracy and precision. Measurements were performed on TQDand QToF mass spectrometers (Waters).

a. Absorption

The absorption and systemic pharmacokinetic behavior of RTA 408 wasstudied in mice, rats, and monkeys following single and repeated (daily)oral administration. Following oral administration of a suspensionformulation at doses of 10 to 100 mg/kg, maximal concentrations wereobserved within 1 to 2 h in mice, and within 1 to 24 h in rats andmonkeys. Systemic exposure to RTA 408 tended to be highest in rats, withlower levels observed in mice and monkeys. Estimates of the apparentterminal half-life of RTA 408 observed after oral administration weregenerally in the 6- to 26-h range, though the apparent prolongedabsorption phase in some instances precluded calculation of a definitivehalf-life estimate.

Systemic exposure to RTA 408 was generally similar in males and females.Exposure to RTA 408 following repeated daily oral administration tendedto be slightly higher (≦2-fold) than the exposure observed after asingle dose. Administration of RTA 408 over a dose range from 3 to 100mg/kg in a suspension formulation generally resulted indose-proportional increases in systemic exposure. However,administration of higher doses (100 to 800 mg/kg in monkeys; 500 to 2000mg/kg in rats) did not result in similar increases in exposure,suggesting saturation of absorption at doses above 100 mg/kg. Followingoral administration of an unoptimized (loose-filled) capsule formulationof RTA 408 (3 mg/kg) to monkeys, dose-normalized systemic exposuretended to be somewhat lower than that observed with a suspensionformulation.

The absorption and systemic pharmacokinetic behavior of RTA 408 wasstudied in rats using single and repeated topical administration. Theadministration of RTA 408 over a range of 0.01% to 3% showed lowerplasma concentrations relative to similar oral dosing. The systemicexposure to RTA 408 generally increased in a dose dependent manner. Thetopical administration was formulated as a suspension in sesame oil.

Using rabbits, the ocular absorption and systemic pharmacokineticbehavior of RTA 408 was evaluated. RTA 408 was administered topically tothe eye once per day for five days. The ocular administration showedlower plasma concentration of RTA 408 relative to when RTA 408 isadministered orally (FIG. 36). The amount of RTA 408 in the plasma evenafter five consecutive days showed only a small change compared to theconcentration after the first dose relative to when RTA 408 wasadministered orally, where plasma concentrations were almost 100-foldhigher (FIG. 36).

b. Distribution

Plasma protein binding of RTA 408 was evaluated in mouse, rat, rabbit,dog, minipig, monkey, and human plasma at RTA 408 concentrations of10-2000 ng/mL using ultracentrifugation methodology. RTA 408 wasextensively bound to plasma proteins. Plasma protein binding in thenonclinical species ranged from 93% (mouse) to >99% (minipig), withbinding of 95% in the toxicology species (rat and monkey) and 97% inhuman. There was no evidence of concentration-dependent protein bindingin any species tested. Results from blood-to-plasma partitioningexperiments indicate that RTA 408 tended to distribute primarily in theplasma fraction of blood in a linear manner, with blood:plasma ratios<1.0 for all species and all concentrations tested.

The distribution of RTA 408 into tissues has been investigated afteroral administration to mice, rats, and monkeys. In the 14-day non-GLPtoxicity studies, select tissues (liver, lung, and brain) were collectedat a single time point (4 h for rat and mouse; 24 h for monkey) afterthe final dose of the study was administered and were analyzed for RTA408 content using LC/MS/MS. RTA 408 readily distributes into lung,liver, and brain. In lung, RTA 408 concentrations at 4 h in mice andrats were similar to or slightly higher (<2-fold) than concentrations inplasma, while at 24 h in monkeys, RTA 408 concentrations in lung were 6-to 16-fold higher than plasma concentrations. A similar pattern wasobserved for brain. In contrast, RTA 408 concentrations in liver were 5-to 17-fold higher than plasma for mice and rats at 4 h, and 2- to 5-foldhigher than plasma at 24 h in monkeys.

The pharmacodynamic effects of RTA 408 in tissues were assessed in mice,rats, and monkeys, by monitoring the induction of Nrf2 target genes inthe same tissues collected for drug exposure from the 14-day toxicitystudies. Induction of Nrf2 target genes by RTA 408 resulted in increasesin the antioxidant response as manifested by dose-dependent increases inNQO1, glutathione S-transferase (Gst), and glutathione reductase (Gsr)enzyme activity in the examined tissues. Enzyme activities were measuredas described above. Furthermore, in rodents, RTA 408 liver contentcorrelated with the level of enzyme activity for NQO1, the prototypicaltarget gene for Nrf2. In monkeys, the level of mRNA expression inperipheral blood mononuclear cells (PBMCs) for both NQO1 andsulfuredoxin 1 (SRXN1) correlated with plasma exposure of RTA 408 (FIGS.37 a & b). Overall, RTA 408 induced biomarkers of Nrf2 in rodents andmonkeys, and such inductions generally correlated well with tissue andplasma exposure to RTA 408.

When RTA 408 was administered to rabbits via ocular topicaladministration, the highest concentrations of the compound were found inthe cornea, retina, or iris while the vitreous humor, aqueous humor, andplasma showed significantly lower concentrations of RTA 408 (FIG. 38).

c. Metabolism

The metabolism of RTA 408 has been investigated after in vitroincubation of RTA 408 for 60 min with liver microsomes from mice, rats,monkeys, and humans in the presence of a nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system and a uridinediphosphate glucuronosyltransferase (UGT) reaction mixture. Extensiveturnover of RTA 408 was observed with primate microsomes, with <10% ofthe parent molecule remaining at the end of the 60-min incubation inboth monkey and human microsomes. In contrast, the extent of metabolismwas lower in rodent microsomes, with >65% of the parent moleculeremaining at the end of the incubation. The lack of available authenticstandards for the various potential metabolites of RTA 408 precludedquantitative evaluation of the observed metabolites. From a qualitativeperspective, a similar pattern of RTA 408 metabolites was observedacross species, and included peaks with masses consistent with reductionand hydroxylation of RTA 408 as well as glucuronidation of RTA 408 or ofits reduction/hydroxylation metabolites. No unique human metaboliteswere observed, with all peaks in the human microsome incubations alsobeing observed in one or more of the preclinical species. In particular,based on in vitro microsome data, all human metabolites were present inrat or monkey, the selected rodent and non-rodent toxicity species.

d. Pharmacokinetic Drug Interactions

The potential for RTA 408 to inhibit cytochrome P450 (CYP450)-mediatedmetabolism was evaluated using pooled human liver microsomes andstandard substrates for specific CYP450 enzymes. RTA 408 directlyinhibited CYP2C8 and CYP3A4/5 with K_(i) values of approximately 0.5 μMfor each enzyme. No meaningful inhibition was observed for the otherenzymes tested (CYP1A2, CYP2B6, CYP2C9, CYP2C19, or CYP2D6), withinhibition <50% at the highest concentration tested (3 μM). In addition,there was little or no evidence of metabolism-dependent inhibition ofany of the enzymes tested. Future studies investigating the potentialfor CYP3A4/5-mediated drug-drug interactions may be warranted based onthese data, and the potentially high concentrations that may be achievedlocally in the gastrointestinal (GI) tract after oral administration.

The potential for RTA 408 to induce CYP450 enzyme expression wasevaluated using cultured human hepatocytes. Under conditions whereprototypical inducers caused the expected increases in CYP activity, RTA408 (up to 3 μM) was not an inducer of CYP1A2, CYP2B6, or CYP3A4 enzymeactivity in cultured human hepatocytes. Enzyme activity was measured bymonitoring substrate conversion of phenacetin, bupropion, andtestosterone for CYP1A2, CYP2B6, and CYP3A4, respectively, in isolatedmicrosomes.

F. Effects of RTA 408 on Acute Radiation Dermatitis

The effects of RTA 408 as a topical or oral preventative for acuteradiation dermatitis have been examined. Using male BALB/c mice, a 30 Gydose of radiation was administered on day 0 (Table 3). The sesame oilvehicle or RTA 408 was administered to the rats on day −5 to −1 and days1 to 30. RTA 408 was administered both orally in 3, 10, and 30 mg/kg insesame oil and topically in percentage composition of 0.01%, 0.1%, and1% in sesame oil. The dermatitis was blindly evaluated every other dayfrom day 4 to day 30. On day 12, the typical peak of dermatitis wasobserved and 4 mice were sacrificed 4 hours after administration of thedose. The remaining mice were sacrificed on day 30 at 4 h post-dose.Plasma was collected on days 12 and 30 as well as irradiated skinsamples for mRNA and histological examination.

TABLE 3 Study Design for Acute Radiation Dermatitis Model Number ofRadiation Treatment Group Animals (Day 0) Treatment Schedule 1  9 males— Untreated — 2 10 males 30 Gy Untreated — 3 14 males 30 Gy VehicleControl Day −5 to −1 & (sesame oil) Day 1 to 30 4 14 males 30 Gy RTA408 - 0.01% Day −5 to −1 & or 3 mg/kg Day 1 to 30 5 14 males 30 Gy RTA408 - 0.1% or Day −5 to −1 & 10 mg/kg Day 1 to 30 6 14 males 30 Gy RTA408 - 1% or Day −5 to −1 & 30 mg/kg Day 1 to 30

In the test groups where the mice were treated with RTA 408, theincidence of dermatitis appeared to be slightly diminished in severitywhen RTA 408 was given in either an oral or topical administration(FIGS. 39-42). Furthermore, curves plotting the average dermatitisclinic score for the test groups as a function of time show some changewith the administration of RTA 408 either in oral or topical form fromthe untreated test groups (FIGS. 43-45) particularly in the case whereRTA 408 was given through an oral administration. Furthermore, as can beseen in Tables 4 and 5 below, the percentage of mice suffering fromdermatitis with a clinical score above 3 was significantly lower formice treated with RTA 408 through an oral administration while thepercentage of mice suffering from dermatitis with a clinical score above2 was slightly lower for test groups who were given a topicaladministration of RTA 408.

TABLE 4 Percentage of mice per testing group which scored above 2 intheir clinical dermatitis exam and given a topical treatment containingRTA 408 % animal- % animal- Day 12 Day 14 Day 16 Day 18 Day 20 Day 22Day 24 Day 26 Day 28 Day 30 days >=2 days >=3 1 no radiation, untreated0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2 irradiated, untreated0.0 50.0 83.3 83.3 83.3 100.0 66.7 50.0 50.0 50.0 35.6 0.0 3 irradiated,sesame oil 21.4 45.0 60.0 50.0 40.0 40.0 0.0 0.0 0.0 0.0 16.6 0.0 4irradiated, RTA 0.0 0.0 20.0 50.0 10.0 40.0 40.0 40.0 20.0 10.0 14.4 0.0408-0.01% 5 irradiated, RTA 7.1 10.0 20.0 80.0 60.0 40.0 30.0 10.0 0.00.0 16.3 0.0 408-0.1% 6 irradiated, RTA 10.7 20.0 10.0 70.0 30.0 10.00.0 0.0 0.0 0.0 9.7 0.0 408-1.0%

TABLE 5 Percentage of mice per testing group which scored above 3 intheir clinical dermatitis exam and given an oral treatment containingRTA 408 % animal- % animal- Day 16 Day 18 Day 20 Day 22 Day 24 Day 26Day 28 days >=2 days >=3 1 no radiation, untreated 0 0 0 0 0 0 0 0.0 0.02 irradiated, untreated 20 40 20 20 20 20 20 39.0 8.8 3 irradiated,sesame oil 35 50 40 30 20 0 0 45.6 10.9 4 irradiated, RTA 408-3 mg/kg 1010 0 0 0 0 0 32.5 1.3 5 irradiated, RTA 408-10 mg/kg 10 25 30 0 0 0 033.8 4.1 6 irradiated, RTA 408-30 mg/kg 10 20 10 0 0 0 0 28.8 2.5

G. Effects of RTA 408 on Fractionated Radiation Dermatitis

Utilizing RTA 408 through topical administration, the effects of RTA 408towards ameliorating the effects of fractionated radiation dermatitiswere measured. Using Balb/c mice, RTA 408 in a topical preparation wasadministered to the mice daily from day −5 to day 30 in three dosesranging from 0.01% to 1%. The mice were irradiated on days 0-2 and 5-7with six 10-Gy doses per day. Clinical dermatitis scores for the micewere evaluated blindly every two days from day 4 until the end of thestudy. In FIG. 46, the graph shows the change in the average clinicalscore for each group were plotted as a function of time. The graph showsa statistically significant improvement in the scores for mice treatedwith 0.1% to 1% topical formulations of RTA 408. Study and treatmentparameters can be found in Table 6.

TABLE 6 Study Conditions for Fractionated Radiation-Induced DermatitisRadiation Number of (Days Treatment Group Animals 0-2, 5-7) TreatmentSchedule 1  9 males — Untreated — 2 14 males 6 × 10 Gy Untreated — 3 18males 6 × 10 Gy Vehicle Control QD Days −5 to 30 (sesame oil) 4 18 males6 × 10 Gy RTA 408 - 0.01% QD Days −5 to 30 5 18 males 6 × 10 Gy RTA408 - 0.1% QD Days −5 to 30 6 18 males 6 × 10 Gy RTA 408 - 1% QD Days −5to 30By analyzing the average clinical scores that were shown in FIG. 46, anarea under the curve (AUC) analysis was performed, which yielded theseverity of the dermatitis relative to how long the dermatitispersisted. This AUC analysis allowed for direct comparison between thedifferent groups of mice and the effect of the different percentagecompositions of RTA 408 (FIG. 47 and Table 7). Administration of topicalRTA 408 formulations reduced Grade 2 and Grade 3 lesions from 60% and33% when the mice were only exposed to the vehicle to 21% and 6% withRTA 408 at 1%, concentration, respectively. The other RTA compositionshowed some activity but was not as significant as that shown by the 1%formulation.

TABLE 7 Percentage of Dermatitis Score for Each Treatment Group Group %Days ≧2 % Days ≧3 No Rad, No Tx 0% 0% Rad, No Tx 66% 31% Rad, Sesame Oil60% 33% Rad, RTA 408 (0.01%) 54% 29% Rad, RTA 408 (0.1%) 40% 13% Rad,RTA 408 (1%) 21% 6%

H. Synergistic Effects of RTA 408 and Cancer Therapeutic Agents on TumorGrowth

A study of the effects of RTA 408 used in combination with traditionalchemotherapeutic agents was carried out to determine the efficacy of thepotential treatment. In vitro studies were carried out to determine theeffects of RTA 408 on two different prostate cancer cell lines, LNCaPand DU-145. As can be seen in FIG. 48 a, the treatment of the prostatecancer cell lines (LNCaP) in vitro with 5-fluorouracil shows astatistically significant increase in cytotoxicity when combined withRTA 408 at doses from ranging from 0.125 to 0.5 μM. Using the prostatecell line DU-145 and docetaxel, RTA 408 amplified the cytotoxicity ofthe chemotherapeutic agent in a statistically significant fashion fordosing of RTA 408 from 0.125 to 0.75 μM as shown in FIG. 48 b. Thisevidence supports the concept that RTA 408 could act synergisticallywith cancer therapeutic agents and may be used in some embodiments toprovide greater efficacy in treating cancer patients.

After the successful results of the in vitro assay, a pilot in vivoassay was carried out using LNCaP/C4-2B and DU145 human prostate cancerengineered to express firefly luciferase (hereafter referred to asC4-2B-Luc and DU145-Luc, respectively). Of note, both of these celllines grow in an androgen-independent fashion. Cells were cultured inRPMI 1640 supplemented with 10% FBS. Cells were harvested using TrypLEExpress (Invitrogen) and washed in PBS and counted. Cells werereconstituted in PBS to arrive at a final concentration of 3×10⁶ cellsper 30 μL (unless otherwise stated) and aliquoted in separate tubes.Growth factor-reduced Matrigel (BD Bioscience) was thawed overnight at+4° C. and transferred into the tubes in 30 μL aliquots. Thecell/Matrigel solutions were transferred to the vivarium and mixed rightbefore injection at a 1:1 ratio. Each mouse (n=1 per group for a totalof three animals) received a single subcutaneous injection of the tumorcells. Tumors were pre-established for 4 weeks. Then, one animal wastreated with RTA 408 (17.5 mg/kg, i.p.) once a day for 3 days (Days −3to −1). On the following day (Day 0), the RTA 408 treated animals andone other animal were treated with a single dose of 18 Gy IR, localizedto the pelvic region where the tumors were implanted. The mouse that waspre-treated with RTA 408 received three additional doses of RTA 408(17.5 mg/kg, i.p.), once every other day, over the following week. Thethird animal received no treatment and served as a positive control.Tumor progression was monitored weekly via live imaging. To detectluciferase-expressing tumor cells, mice were IP injected withD-Luciferin 5 min prior to imaging according to the manufacturer'sprotocol (Caliper LifeScience). Prior to imaging mice were anesthetizedby isoflurane inhalation and imaged on the IVIS Lumina XR system(Caliper LifeScience). For standardization, minimal exposure timenecessary to image control tumor was determined and all animals wereimaged under these conditions. On Day 7, no apparent reduction in tumorsize was visible in the IR treated animal compared to the control,whereas the animal receiving both RTA 408 and IR showed a smaller tumorimage. On Day 14 and Day 21, the control animal showed continued tumordevelopment and growth while the animal treated with ionizing radiationshowed some improvement, most notably at Day 21. On the other hand, theanimal treated with RTA 408 and ionizing radiation showed no progressionfrom Day 7 to Day 14 and had no visible tumor on Day 21. The progress ofthe tumor per week can be seen in FIG. 49. Both the in vitro and in vivodata show that RTA 408 appears to complement the activity of differentcancer therapeutic agents thus increasing the agent's efficacy.

I. Effects of RTA 408 on a Model of Ocular Inflammation

A study of the effects of RTA 408 on ocular inflammation was carried outusing rabbits of the New Zealand albino strain. The rabbits were dividedinto 5 groups of 12 rabbits which were given three differentconcentrations of RTA 408 (0.01%, 0.1%, and 1%), Voltarene© collyre at0.1% and the vehicle (sesame oil). Each rabbit was given threeinstillations within 60 min before induction of paracentesis and twoinstillations within 30 min after induction of paracentesis. Eachinstillation was 50 μL and given in both eyes. Aqueous humor for 6animals per time-point was collected 30 min and again 2 h afterinduction of paracentesis. The amount of inflammation was determined byprotein concentration in the aqueous humor. As shown in FIG. 50, RTA 408showed a reduction in aqueous humor protein similar to that of thehighest concentration of any of the other reference compounds (MaxiDexor mapracorat) at only 0.01% RTA 408 in the formulation. The effects ofincreasing concentration of RTA 408 appeared to be negligible as allconcentrations of RTA 408 appeared to show relatively similar effectswithin error in reducing aqueous humor protein concentration.

J. Polymorph Screen

A preformulation and polymorphism study was performed for compound63415. As part of this study, a preliminary polymorphism program wascarried out with the aim to identify the most stable anhydrous form atroom temperature and possible hydrates with a reasonably highprobability. A total of 30 crystallization experiments, including phaseequilibrations, drying experiments and other techniques, were carriedout. All obtained solids were characterized by FT-Raman spectroscopy.All new forms were characterized by PXRD and TG-FTIR, and optionally byDSC and DVS.

In addition, the amorphous form was prepared and characterized. Severalexperiments using different techniques and approaches were carried outto prepare the amorphous form. The amorphous form was characterized byFT-Raman spectroscopy, PXRD, TG-FTIR, DSC, DVS, and Karl-Fischertitration. The stability of the amorphous form was tested at elevatedhumidity and temperature conditions over the course of four weeks.

1. Starting Material and Nomenclature

Two batches of 63415 were used as starting materials (Table 8). 63415 isalso referred to as PP415 in this disclosure. All samples received orgenerated during this project received a unique identification code ofthe form PP415-Px, where Px refers to the sample/experiment number (x=1,2, . . . , n).

TABLE 8 Starting materials Sample Material Amount Received PP415-P163415, batch #: 0141-66-1; 5.0 g Mar. 25, 2011 MW = 554.7 g/mol,C₃₃H₄₄F₂N₂O₃ PP415-P40 63415, batch #: 2083-69-DC; 5.0 g May 27, 2011 MW= 554.7 g/mol, C₃₃H₄₄F₂N₂O₃

2. Compound 63415, batch #0414-66-1 (PP415-P1): The Amorphous Form

The 63415, batch #0414-66-1, starting material was characterized byFT-Raman spectroscopy, PXRD, TG-FTIR, Karl-Fischer titration, ¹H-NMR,DSC, DVS, and approximate solubility measurements. The results aresummarized in Table 9.

TABLE 9 Characterization of the 63415 starting material (PP415-P1)Method Results FT-Raman will be used as the reference PXRD no sharp peakpattern, material is amorphous TG-FTIR ~0.9 wt.-% (~0.1 eq.) EtOH withtraces of H₂O from 25° C. to 200° C., decomposition at T > 290° C. Karl-0.5 wt.-% H₂O Fischer ¹H-NMR agrees with structure, ~0.08 eq. EtOH DSC1^(st) heating scan: glass transition T_(g) = 152.7° C. (ΔCp = 0.72 J/g°C.); 2^(nd) heating scan: glass transition T_(g) = 149.7° C. (ΔCp = 0.45J/g° C.) DVS slightly hygroscopic; Δm = +0.4% (50%→85% r.h.); FT-Ramanand PXRD unchanged

The FT-Raman spectrum (FIG. 58) will be used as the reference spectrumfor the starting material. PXRD (FIG. 59) shows no sharp peak pattern.The broad halo at ˜10-20°2θ is characteristic for amorphous materials.

The TG-FTIR thermogram (FIG. 60) shows the gradual loss of ˜0.9 wt.-%EtOH (i.e., ˜0.1 eq.) with traces of H₂O between 25 and 200° C.Decomposition starts at T>290° C.

A water content of 0.5 wt.-% was determined by Karl-Fischer titration.

The ¹H-NMR spectrum (FIG. 61) agrees with the structure and shows ˜0.08eq. EtOH, in agreement with the TG-FTIR thermogram.

The DSC thermogram (FIG. 62) shows in a first heating scan a glasstransition of the amorphous material at T_(g)=152.7° C. (ΔC_(p)=0.72J/g° C.). In a second scan after quench cooling, the glass transitionoccurs at T_(g)=149.7° C. (ΔC_(p)=0.45 J/g° C.).

The DVS isotherm (FIG. 63) shows that a gradual mass loss of 1.0 wt.-%occurred upon lowering the relative humidity from 50% r.h. to 0% r.h.;equilibrium was reached at 0% r.h. Upon increasing the relative humidityto 95% r.h. a gradual mass gain of 2.1 wt.-% (relative to the mass at 0%r.h.) occurred; equilibrium was reached at 95% r.h. Upon lowering therelative humidity from 95% r.h. to 50% r.h. the final mass was 0.2 wt.-%below the starting mass. The mass increase of 0.4 wt.-% at 85% r.h.(relative to the starting mass) classifies the sample as slightlyhygroscopic.

The FT-Raman spectrum (FIG. 64) and PXRD pattern (FIG. 65) of the sampleafter the DVS measurement are unchanged compared to the spectrum andpattern of the sample before the measurement.

The approximate solubility of the PP415-P1 starting material wasmeasured in twelve solvents and four solvent mixtures at r.t. by manualdilution combined with visual observation (Table 10). Due to theexperimental error inherent in this method, the solubility values areintended to be regarded as rough estimates and are to be used solely forthe design of crystallization experiments. All solvent mixtures arelisted as ratios by volume (v/v).

TABLE 10 Approximate solubility of the PP415-P1 (amorphous) startingmaterial Solvent Solubility S [mg/mL] toluene S > 200 DCM S > 200 EtOAcS > 210 acetone S > 230 MeCN S > 230 DMF S > 210 MeOH S < 210 EtOH^(a)105 < S < 210 2PrOH 16 < S < 19 DEE S ≧ 1^(d )  heptane S < 1  H₂O S <1  2PrOH/H₂O (9:1)^(b) 7.9 < S < 8.5 MeCN/H₂O (2:3)^(c) S < 1 EtOAc/heptane (1:1)^(a) 100 < S < 200 toluene/DEE (1:1)^(a) S > 220^(a)observed precipitation after ~1 d; ^(b)water activity a(H₂O) ~0.7 at25° C.; ^(c)water activity a(H₂O) > 0.9 at 50° C.; ^(d)incompletedissolution at first (S < 1), but solid residue dissolved completelyovernight (S > 1).

3. Compound 63415, batch #2083-69-DC (PP415-P40): Class 2

63415, batch #2083-69-DC, is a heptane solvate. This material(PP415-P40) was characterized by PXRD and found to correspond to class 2(FIG. 66).

Class 2 likely corresponds to isostructural, non-stoichiometric (<0.5eq.) solvates (of heptane, cyclohexane, isopropyl ether, 1-butanol,triethyl amine, and possibly other solvents, such as hexane and otherethers) with tightly bound solvent.

The small peaks visible in the pattern of PP415-P40 at 7.9°2θ and13.8°2θ do not correspond to peaks of classes 3, 4, or 5. Their originis not clear at this point.

4. Chemical Stability of the Amorphous Form

The chemical stability of the amorphous form was investigated indifferent solvents over the course of seven days.

Solutions/suspensions with a concentration of 1 mg/mL were prepared infour organic solvents (acetone, MeOH, MeCN, EtOAc) and three aqueoussurfactant media (1% aq. SDS, 1% aq. Tween 80, 1% aq. CTAB).

Four separate solutions/suspensions were prepared for each solvent,equilibrated for 6 h, 24 h, 2 d, and 7 d and subsequently analyzed byHPLC.

The relative area-% obtained from the HPLC chromatograms are given inTable 11. The compound seems to be somewhat unstable in the diluent(0.1% formic acid in MeCN); over the course of the sequence (i.e.,within ˜24 hours) the area-% of a reference sample (PP415-P1, ran at thebeginning and the end of the sequence) decreased from 99.9% to 99.3% at254 nm and from 99.9% to 99.5% at 242 nm. Due to this effect, thesamples measured towards the end of the sequence (set up in thefollowing order: 7 d, 2 d, 24 h, 6 h), might be affected and theobtained area-% might be underestimated.

TABLE 11 Chemical stability experiments with the amorphous form of 63415(PP415-P1)^(a) at 254 nm at 242 nm Solvent 7 d 2 d 24 h 6 h 7 d 2 d 24 h6 h acetone 99.6% 99.6% 99.6% 99.6% 99.7% 99.7% 99.6% 99.6% EtOAc 99.8%99.8% 99.8% 99.7% 99.8% 99.9% 99.8% 99.7% MeOH 99.8% 99.8% 99.7% 99.8%99.8% 99.9% 99.7% 99.8% MeCN 97.7% 99.5% 99.3% 99.4% 97.4% 99.5% 99.3%99.3% Tween 1%^(b) 97.7% 97.1% 95.1% 97.6% 98.7% 98.7% 96.2% 99.1% SDS1%^(c) 99.7% 99.6% 99.7% 99.7% 99.8% 99.7% 99.7% 99.7% CTAB 1% 99.3%99.4% 99.4% 99.6% 99.3% 99.4% 99.4% 99.7% ^(a)at the third wavelength(210 nm), the signal intensity was weak and the signal-to-noise ratiolarge, thus integration was not carried out ^(b)suspensions, not allmaterial dissolved for all time points ^(c)suspensions, not all soliddissolved for time points 24 h and 6 h

Decomposition ≧1% was observed for solutions in MeCN after seven daysand for suspensions in the 1% aqueous Tween 80 media (at all time pointsat 254 nm and after 24 h, 2d, and 7d at 242 nm).

5. Storage Stability of the Amorphous Form

To learn more about its fundamental properties and physical stability,the amorphous form of 63415 was stressed by storage at elevatedtemperatures and relative humidities.

Samples of the amorphous form (the PP415-P 1 starting material) werestored open at 25° C./˜62% r.h. (over saturated aqueous solution ofNH₄NO₃) and 40° C./˜75% r.h. (over saturated aqueous solution of NaCl)and closed at 60° C. and 80° C. (Table 12). At time points 0 w, 1 w, 2w, and 4 w the samples were examined by PXRD and compared to thestarting material, PP415-P1.

TABLE 12 Storage stability experiments with the amorphous form of 63415(PP415-P1) Sample Conditions Time Point PXRD Result PP415-P2a open, 25°C./~62% r.h. 1 w amorphous PP415-P2b open, 25° C./~62% r.h. 2 wamorphous PP415-P2c open, 25° C./~62% r.h. 4 w amorphous PP415-P3a open,40° C./~75% r.h. 1 w amorphous PP415-P3b open, 40° C./~75% r.h. 2 wamorphous PP415-P3c open, 40° C./~75% r.h. 4 w amorphous PP415-P4aclosed, 60° C. 1 w amorphous PP415-P4b closed, 60° C. 2 w amorphousPP415-P4c closed, 60° C. 4 w amorphous PP415-P5a closed, 80° C. 1 wamorphous PP415-P5b closed, 80° C. 2 w amorphous PP415-P5c closed, 80°C. 4 w amorphous

After one week (time point 1 w, FIG. 67), two weeks (time point 2 w,FIG. 68), and four weeks (time point 4 w, FIG. 69) all four samples werestill amorphous, as the powder X-ray diffractograms show no differencescompared to the starting material at time point 0 w.

6. Crystallization and Drying Experiments

a. Crystallization Experiments

Phase equilibrations, crystallizations from hot solutions, andevaporation experiments were carried out starting from the amorphousform in order to identify with reasonably high probability the moststable anhydrous form at r.t. and possible hydrates. All obtainedmaterials were characterized by FT-Raman spectroscopy; selected sampleswere also characterized by PXRD.

The FT-Raman spectra were grouped into classes according to thesimilarity of their peak positions. The original sample (PP415-P1, seeTable 8) was classified along with the crystallization products. Thespectra within a class, however, are not strictly identical, butsimilar. Small differences and peak shifts might exist. Considering theFT-Raman spectra alone, it is difficult to determine if the spectra ofone class belong to the same polymorphic form.

The peaks in the PXRD patterns were determined and the patterns thenclassified into clusters using the PANalytical X′Pert (Highscore Plus)software. These clusters identify patterns of a high similarity.However, small but significant differences exist within a cluster. Thus,the patterns within a cluster do not necessarily correspond to the samepolymorphs, but represent different forms with very similar molecularstructures. The FT-Raman classes correspond in all cases to the PXRDclusters.

b. Suspension Equilibration Experiments

Suspension equilibration experiments were carried out in one solvent andeleven solvent mixtures (Table 13). Suspensions of ˜100 mg of PP415-P1in 0.2-2.0 mL of the selected solvents were prepared and shaken for 4-15days at 22-24° C. The solids were recovered and characterized byFT-Raman spectroscopy; most were characterized also by PXRD.

TABLE 13 Suspension equilibration experiments starting from theamorphous form (PP415-P1) FT-Raman PXRD Sample Solvent/Mixture classcluster PP415-P6 2PrOH 3 3 PP415-P7 1:2 EtOAc/heptane 2 2 PP415-P8 1:2acetone/hexane 2 2 PP415-P9 1:3 toluene/DEE  2^(d) — PP415-P10 1:3MeOH/TBME 2 2 PP415-P11 1:2 MEK/cyclohexane  2^(d) — PP415-P12 9:1EtOH/H₂O^(a) 3 3 PP415-P13 7:3 MeCN/H₂O^(b)  4^(d) 4 PP415-P14 ~1:1THF/H₂O^(c)  5^(d) 5 PP415-P29 1:2 EtOAc/TEA 2 2 PP415-P31 9:1 PEG/H₂O 11 PP415-P35 7:3 MeCN/H₂O^(b)  4^(d) 4 water activities: ^(a)a(H₂O) ~0.5at 50° C.; ^(b)a(H₂O) ~0.85 at 50° C.; ^(c)a(H₂O) > 0.99 at 64° C.;^(d)the spectrum contains solvent signals

c. Crystallizations from Hot Solutions

Hot solutions of PP415-P1 were prepared in one solvent and four solventmixtures (Table 14). Upon slow cooling to 5° C. at a rate of ˜0.2 K/min,precipitation was observed in three cases (-P20, -P21, -P24). In twocases (-P22, -P23) no solid precipitated, even after storage at 4-5° C.for two days. Here, the solvent was evaporated under N₂ flow at r.t. Thesolids were recovered and characterized by FT-Raman spectroscopy and forthose with spectra different from the amorphous starting material,FT-Raman class 1, also by PXRD.

TABLE 14 Slow cooling experiments starting from the amorphous form(PP415-P1) FT-Raman PXRD Sample Solvent/Mixture Conditions class clusterPP415-P20 ~2:1 acetone/H₂O 55° C. → 5° C. 3^(b) 3 PP415-P21 ~1:5 EtOH/75° C. → 5° C. 2  2 cyclohexane PP415-P22 ~1:3 MeCN/ 75° C. → 5° C.^(a)1^(b) — toluene PP415-P23  1:3 EtOAc/ 75° C. → 5° C.^(a) 1^(b) — dioxanePP415-P24 1BuOH 75° C. → 5° C. 2^(b) 2 ^(a)no precipitation after slowcooling and stirring at 5° C. for 2 days; evaporated solvent under N₂flow at r.t. ^(b)the spectrum contains solvent signals

d. Evaporation/Precipitation Experiments

Clear solutions of PP415-P1 were prepared in three solvent mixtures(Table 15). The solvents were then slowly evaporated at r.t. underambient conditions. However, in two of the three experiments (-P15 and-P17) white solid precipitated before evaporation began. The obtainedsolids were examined by FT-Raman spectroscopy and PXRD.

TABLE 15 Slow evaporation experiments with the amorphous form (PP415-P1)Sample Solvent/Mixture FT-Raman class PXRD cluster PP415-P15 1:2 DCM/IPE2^(a) 2 PP415-P16 1:2 MeOH/toluene 1^(a) — PP415-P17 1:3 EtOAc/heptane2^(a) 2 ^(a)the spectrum contains solvent signals

e. Drying Experiments

At least one sample of each class was dried under vacuum with the aim todesolvate the solvates and to obtain non-solvated crystalline forms of63415 (Table 16). The dried materials were characterized further byFT-Raman, PXRD, and TG-FTIR.

TABLE 16 Drying experiments carried out on samples obtained from thecrystallization experiments Starting Material Sample (Class) ConditionsResult PP415-P18 PP415-P15 (2) r.t., 2-10 mbar, ~2 h 2  PP415-P19PP415-P17 (2) r.t., 2-10 mbar, ~2 h 2  PP415-P25 PP415-P6 (3) r.t., ~3mbar, ~5 d; 3  60° C., 5-10 mbar, 2 × 1 h; 40-50° C., 5-20 mbar, ~1 dPP415-P26 PP415-P13 (4) r.t., ~3 mbar, ~5 d; 4  60° C., 5-10 mbar, 2 × 1h; 40-50° C., 5-20 mbar, ~1 d PP415-P27 PP415-P14 (5) r.t., ~3 mbar, ~5d; 1^(a) 60° C., 5-10 mbar, 2 × 1 h; 40-50° C., 5-20 mbar, ~1 dPP415-P28 PP415-P21 (2) r.t., ~3 mbar, ~5 d; 2^(b) 60° C., 5-10 mbar, 2× 1 h; 40-50° C., 5-20 mbar, ~1 d PP415-P30 PP415-P7 (2) 50-70° C., 1-10mbar, 3 d 2  PP415-P32 PP415-P19 (2) 80° C., <1 × 10⁻³ mbar, 3 d 2^(b)PP415-P33 PP415-P25 (3) 80° C., <1 × 10⁻³ mbar, 3 d 3  PP415-P34PP415-P28 (2) 80° C., <1 × 10⁻³ mbar, 3 d 2^(b) PP415-P36 PP415-P35 (4)80° C., <1 × 10⁻³ mbar, 3 d 4^(c) PP415-P37 PP415-P35 (4) 80° C., N₂flow, 3 d 4^(c) PP415-P44a PP415-P41 (5) 80° C., 100 mbar, 2 d 1^(a)PP415-P46a PP415-P45 (6) 80° C., 100 mbar, 4 d 1^(a) ^(a)desolvationsuccessful, significant reduction of solvent content, sample mainlyamorphous; only few, broad peaks in PXRD ^(b)sample less crystalline, asindicated by broader peaks in PXRD ^(c)desolvation successful,significant reduction of solvent content, sample still crystalline; nochange in structure

7. Characterization of New Forms (Classes)

a. Summary of New Classes

In addition to the amorphous form of 63415, four new crystalline formswere obtained in this study (Table 17).

TABLE 17 Summary of obtained classes Result of Class CharacteristicsDrying Experiments Class 1 amorphous form — Class 2 isostructuralsolvates (e.g., heptane) drying unsuccessful Class 3 isostructuralsolvates (e.g., ethanol) drying unsuccessful Class 4 MeCN solvate &desolvated solvate drying successful, structure unchanged Class 5 THFsolvate drying resulted in amorphous form

Class 2: Most crystallization experiments resulted in solid material ofclass 2. These samples likely correspond to isostructural,non-stoichiometric (<0.5 eq.) solvates (of heptane, cyclohexane,isopropyl ether, 1-butanol, triethylamine, and possibly hexane and otherethers, etc.) with tightly bound solvent molecules. The Raman spectraand PXRD patterns within this class are very similar to each other, thusthe structures might be essentially identical with only smalldifferences due to the different solvents that were incorporated.

Drying experiments on class 2 samples have not resulted in acrystalline, non-solvated form. Even elevated temperatures (80° C.) anda high vacuum (<1×10⁻³ mbar) could not remove the tightly bound solventmolecules completely; a solvent content of >2 wt.-% always remained. Thecrystallinity of these samples is reduced, but neither transformationinto a different structure nor substantial amorphization was observed.

Class 3: Solid material of class 3 was obtained from severalcrystallization experiments. The samples of class 3 are likelyisostructural solvates of 2PrOH, EtOH, and probably acetone with tightlybound solvent molecules. They could correspond to either stoichiometrichemisolvates or non-stoichiometric solvates with a solvent content of˜0.5 eq. As with class 2, the Raman spectra and PXRD patterns withinthis class are very similar to each other, indicating similar structuresthat incorporate different solvents. Similar to class 2, dryingexperiments were also unsuccessful. The very tightly bound solventmolecules could only partially be removed (˜5.4 wt.-% to ˜4.8 wt.-%, upto 3 d at 1×10⁻³ mbar and 80° C.). The PXRD pattern remained unchanged.

Class 4: Material of class 4 was only obtained from a 7:3 MeCN/H₂Osolvent system. It most likely corresponds to a crystalline acetonitrilehemisolvate.

By drying (under vacuum or N₂ flow at elevated temperatures) most of thesolvent could be removed without changing or destroying the crystalstructure (PXRD remained unchanged). Thus, a crystalline, non-solvatedform (or rather desolvated solvate) was obtained. It is slightlyhygroscopic (mass gain of ˜0.7 wt.-% from 50% r.h. to 85% r.h.) and hasa possible melting point at 196.1° C. (ΔH=29.31 J/g).

Class 5: Class 5 was also obtained from only one solvent system (˜1:1THF/H₂O) and contains bound THF (and maybe H₂O). As the content of thetwo components cannot be quantified separately, the exact nature of thiscrystalline solvate cannot be determined.

Drying of class 5 resulted in complete desolvation and transformationinto the amorphous form (class 1). One possible process to prepare theamorphous form from class 2 material is a transformation of class 2 toclass 5, followed by drying and amorphization.

b. Class 1—the Amorphous Form

Class 1, the amorphous form of 63415, was obtained from a fewcrystallization experiments (Table 18). Most crystallization experimentsresulted in crystalline material of classes 2, 3, 4, or 5.

The starting material, PP415-P1, is amorphous and belongs to class 1.Further experiments, exclusively aimed at preparing the amorphous form(class 1), were carried out.

TABLE 18 Crystallization experiments resulting in solid material ofclass 1 Character- Sample Method Solvent ization Drying PP415-P31 susp.equil. 9:1 PEG/H₂O Raman, — PXRD PP415-P22 slow ~1:3 MeCN/toluene vis.obs., — cooling^(a) Raman PP415-P23 slow cooling 1:3 EtOAc/dioxane vis.obs., — Raman PP415-P16 evap./precip. 1:2 MeOH/toluene vis. obs., —Raman ^(a)no precipitation after slow cooling and stirring at 5° C. for2 days; evaporated solvent under N₂ flow at r.t.

c. Class 2—Isostructural Solvates (e.g., Heptane)

Most crystallization experiments resulted in solid material of class 2(Table 19). In addition, one batch of a class 2 heptane solvate,PP415-P40, was used as starting material (see Table 8).

The FT-Raman spectra of class 2 are clearly similar to each other (FIG.70) but show small differences. They differ significantly from thespectrum of the amorphous starting material, class 1 (FIG. 71) and fromthe spectra of classes 3, 4, and 5 (FIG. 72).

The PXRD patterns of class 2 (FIG. 73) confirm the crystallinity of thematerials. The patterns of the samples are very similar to each otherbut show small differences (FIG. 74). The class 2 patterns differclearly from the patterns of classes 3, 4, and 5 (FIG. 75).

The TG-FTIR thermogram of sample PP415-P7 (FIG. 76) shows the loss of˜7.5 wt.-% EtOAc and heptane in two steps from ˜100° C. to 290° C. anddecomposition at temperatures T>290° C. Before the TG-FTIR experiments,the samples were dried briefly (for ˜5 min) under vacuum (10-20 mbar) toremove excess, unbound solvent. The loss of both EtOAc and heptane occurtogether in the same temperature range; both solvents seem tightly boundwithin the structure. The theoretical EtOAc content (b.p.=76° C.) of ahemisolvate is 7.4 wt.-%, the theoretical heptane content (b.p.=98° C.)of a hemisolvate is 8.3 wt.-%. Unfortunately, the content of the twocomponents cannot be quantified separately.

The TG-FTIR thermogram of sample PP415-P21 (FIG. 77) shows the loss of˜5.8 wt.-% cyclohexane in two steps from ˜140° C. to ˜250° C. anddecomposition at temperatures T>250° C. With the boiling point ofcyclohexane at 81° C., the solvent seems tightly bound within thestructure. The theoretical cyclohexane content of a hemisolvate is 7.1wt.-%. Thus, sample PP415-P18 possibly corresponds to anon-stoichiometric cyclohexane solvate (with <0.5 eq. solvent content).

The TG-FTIR thermogram of sample PP415-P24 (FIG. 78) shows the loss of˜16.6 wt.-% 1BuOH in a step from ˜50° C. to ˜160° C., further loss of1BuOH (6.6 wt.-%) in a second step from 160° C. to 230° C. anddecomposition at temperatures T>230° C. With the boiling point of 1BuOHat 117° C., the solvent of at least the second step seems tightly boundwithin the structure. The theoretical 1BuOH content of a hemisolvate is6.3 wt.-%.

The TG-FTIR thermogram of sample PP415-P29 (FIG. 79) shows the loss of˜5.1 wt.-% EtOAc and TEA from ˜50° C. to ˜220° C., most of it in a stepfrom 180° C. to 210° C. Decomposition occurs at temperatures T>220° C.The loss of both EtOAc and TEA occur together in the same temperaturerange; both solvents seem tightly bound within the structure (with theboiling point of EtOAc at 77° C. and of TEA at 89° C.).

The TG-FTIR thermogram of sample PP415-P47 (FIG. 80) shows the typicaltwo-step mass loss for class 2 (total of ˜7.9 wt.-% EtOAc) attemperatures up to 240° C., indicating very tightly bound solventmolecules.

The TG-FTIR thermogram of sample PP415-P48 (FIG. 81) shows the mass lossof ˜3.5 wt.-% ethyl formate and water, at first gradually and then in aclear step between 180° C. and 200° C. There might be further loss ofethyl formate concomitant with the decomposition at T>240° C.

Thus, the samples of class 2 might all correspond to non-stoichiometric(<0.5 eq.), isostructural solvates with tightly bound solvent molecules.As the Raman spectra and PXRD patterns within this class are verysimilar to each other, the structures might be essentially identical toeach other with only small distortions of the unit cell dimensions orsmall changes of atomic positions within the unit cell, due to thedifferent sizes and shapes of the incorporated solvent molecules.

TABLE 19 Crystallization experiments resulting in solid material ofclass 2 Sample Method Solvent Characterization Drying PP415-P7 susp.equil. 1:2 EtOAc/heptane Raman, PXRD, TG-FTIR x PP415-P8 susp. equil.1:2 acetone/hexane Raman, PXRD — PP415-P9 susp. equil. 1:3 toluene/DEERaman, PXRD — PP415-P10 susp. equil. 1:3 MeOH/TBME Raman, PXRD —PP415-P11 susp. equil. 1:2 MEK/cyclohexane Raman — PP415-P29 susp.equil. EtOAc/TEA Raman, PXRD, TG-FTIR — PP415-P15 evap./precip. 1:2DCM/IPE Raman, PXRD x PP415-P17 evap./precip. 1:3 EtOAc/heptane Raman,PXRD x PP415-P21 slow cooling ~1:5 EtOH/cyclohexane Raman, PXRD, TG-FTIRx PP415-P24 evap./precip. 1BuOH Raman, PXRD, TG-FTIR — PP415-P43^(a)evaporation (8:2) THF/hexane PXRD — PP415-P47^(a) evaporation EtOAcPXRD, TG-FTIR — PP415-P48^(a) evaporation ethyl formate PXRD, TG-FTIR —^(a)starting material: PP415-P40, class 2; in all other experimentsPP415-P1, class 1, was used as starting material.

d. Drying Experiments on Samples of Class 2

Several samples of class 2 were dried under vacuum (and some at elevatedtemperatures) and in an attempt to desolvate them with the aim to obtainan anhydrous form of 63415. Details and characterizations of the driedsamples are provided below in Table 20.

However, even drying for three days at 80° C. and a vacuum <1×10⁻³ mbarcould not remove the tightly bound solvent molecules completely; asolvent content of >2 wt.-% remained (see samples -P32 and -P34). ThePXRD patterns show a reduced crystallinity of these samples, but notransformation into a different structure was observed.

TABLE 20 Drying experiments on class 2 samples Starting Material DriedMaterial Solvent Drying Solvent Sample Content Conditions Sample ContentClass PP415-P7 EtOAc/heptane 50-70° C., PP415-P30 heptane 2  (~7.5%)1-10 mbar, 3 d (~2.5%) PP415-P15 IPE r.t., 2-10 mbar, PP415-P18 IPE 2 (unknown) ~2 h (~7.0%) PP415-P17 EtOAc(?)/heptane r.t., 2-10 mbar, ~2 hPP415-P19 heptane 2  (unknown) (~7.6%) PP415-P19 heptane 80° C., <1 ×10⁻³ mbar, PP415-P32 heptane 2^(a) (~7.6%) 3 d (~2.2%) PP415-P21cyclohexane r.t., ~3 mbar, ~5 d; PP415-P28 cyclohexane 2^(a) (~5.8%) 60°C., 5-10 mbar, (~3.0%) 2 × 1 h; 40-50° C., 5-20 mbar, ~1 d PP415-P28^(a)cyclohexane 80° C., <1 × 10⁻³ mbar, PP415-P34 cyclohexane 2^(a) (~3.0%)3 d (~2.3%) ^(a)according to PXRD somewhat less crystalline

Thus, the class 2 solvates seem to have very tightly bound solventmolecules. They are difficult to desolvate or transform/amorphize.

e. PP415-P7→PP415-P30

The solid material of sample PP415-P7, class 2, obtained from asuspension equilibration experiment in 1:2 EtOAc/heptane was dried (asPP415-P30) under vacuum for several days (1-10 mbar, 50-70° C.).

The FT-Raman spectrum of the dried class 2 material (PP415-P30) showssmall differences from the original spectrum (sample PP415-P7, FIG. 82)but still corresponds to class 2.

The PXRD pattern of the dried class 2 material (PP415-P30) showsslightly broader, less intense peaks (FIG. 83) but still corresponds toclass 2.

The TG-FTIR thermogram of the dried sample PP415-P30 (FIG. 84) shows theloss of ˜2.5 wt.-% heptane (and some EtOAc) in two steps from ˜50° C. to˜250° C. and decomposition at temperatures T>250° C. Compared to theTG-FTIR of sample PP415-P7 (FIG. 76), the two steps of solvent loss arepreserved, but the total amount of solvent in the sample has decreasedfrom ˜7.5 wt.-% in PP415-P7 to ˜2.5 wt.-% in PP415-P30.

Thus, the attempt to desolvate this solvate at elevated temperatures(50-70° C.) and a vacuum of 1-10 mbar) has caused only a partial loss ofsolvent.

f. PP415-P15→PP415-P18

The solid material of sample PP415-P15, class 2, obtained from aprecipitation experiment in 1:2 DCM/IPE was dried (as PP415-P18) undervacuum (˜2-20 mbar) at r.t. for ˜2 h.

The FT-Raman spectrum of PP415-P18 is identical to the spectrum ofsample PP415-P15 (FIG. 85), both correspond to class 2.

The PXRD pattern of PP415-P18 shows small differences to the pattern ofPP415-P15 (FIG. 86). PP415-P18 still corresponds to class 2.

The TG-FTIR thermogram (FIG. 87) shows the loss of ˜7.0 wt.-% IPE in twosteps from ˜140° C. to ˜250° C. and decomposition at temperatures T>250°C. With the boiling point of IPE being 67° C., the solvent seems tightlybound within the structure. The theoretical IPE content of a hemisolvateis 8.4 wt.-%.

Unfortunately, no TG-FTIR was recorded of the material before the dryingstep. However, as the solvent seems so tightly bound into the structureand no (or only small) changes are observed in the FT-Raman spectra andPXRD patterns, it is assumed that the drying has had no significanteffect on structure or solvent content.

g. PP415-P17→PP415-P19→PP415-P32

The solid material of sample PP415-P17, class 2, obtained from aprecipitation experiment in 1:3 EtOAc/heptane was dried (as PP415-P19)under vacuum (˜2-20 mbar) at r.t. for ˜2 h.

The FT-Raman spectrum of PP415-P19 is identical to the spectrum ofsample PP415-P17 (FIG. 88); no changes can be observed, and bothcorrespond to class 2.

The PXRD pattern of PP415-P19 differs slightly from the pattern ofPP415-P17 (FIG. 89) but still corresponds to class 2.

The TG-FTIR thermogram (FIG. 90) shows the loss of ˜7.6 wt.-% heptane intwo steps from ˜140° C. to ˜270° C. and decomposition at temperaturesT>270° C. With the boiling point of heptane being 98° C., the solventseems tightly bound in the structure. The theoretical heptane content ofa hemisolvate is 8.3 wt.-%.

A further drying experiment (80° C., <1×10⁻³ mbar, 3 days) was carriedout on the same sample as PP415-P32.

The FT-Raman spectrum remained unchanged (FIG. 88). The PXRD patternstill corresponded to class 2 (FIG. 89), but the sample was lesscrystalline (as the peaks were broader and had a lower S/N ratio).

The TG-FTIR thermogram (FIG. 90) shows the loss of ˜2.2 wt.-% heptane,most of it in a step from 170° C. to 200° C. and decomposition attemperatures T>250° C.

Thus, the heptane content was reduced only from 7.6 wt.-% to 2.2 wt.-%,confirming the tight binding of the solvent molecules.

h. PP415-P21→PP415-P28→PP415-P34

The solid material of sample PP415-P21, class 2, obtained from a slowcooling experiment in ˜1:5 EtOH/cyclohexane was dried (as PP415-P28)under vacuum for several days (2-20 mbar, r.t. to 60° C.).

The FT-Raman spectrum of the dried class 2 material (PP415-P28) showssmall differences to the spectrum of class 2 (sample PP415-P21, FIG.92), but still corresponds to class 2.

The PXRD pattern of the dried class 2 material (PP415-P28) showsbroader, less intense peaks compared to the pattern of PP415-P21 (FIG.93), indicating that the dried sample is less crystalline. However, thepattern still corresponds to class 2.

The TG-FTIR thermogram of the dried sample PP415-P28 (FIG. 94) shows theloss of ˜3.0 wt.-% cyclohexane in two steps from ˜140° C. to ˜250° C.and decomposition at temperatures T>250° C. Compared to the TG-FTIR ofsample PP415-P21 (FIG. 77), the two steps of solvent loss are preserved,but the total amount of solvent in the sample has decreased from ˜5.8wt.-% in PP415-P21 to ˜3.0 wt.-% in PP415-P28.

Thus, the desolvation of this solvate seems to have caused only apartial loss of solvent, parallel to a partial loss of crystallinity.

Further drying of this sample (at 80° C., <1×10⁻³ mbar, 3 days) wascarried out as PP415-P34.

The FT-Raman spectrum remained unchanged (FIG. 92). The PXRD patternstill corresponded to class 2 (FIG. 93), but the sample was lesscrystalline (as the peaks were broader and had a lower S/N ratio).

The TG-FTIR thermogram (FIG. 95) shows the loss of ˜2.3 wt.-%cyclohexane, in two steps from 25° C. to 270° C. and decomposition attemperatures T>270° C.

Thus, the cyclohexane content was reduced only from 3.0 wt.-% to 2.3wt.-% confirming the tight binding of the solvent molecules.

i. Class 3—Isostructural Solvates (e.g., Ethanol)

Several crystallization experiments resulted in solid material of class3 and were characterized by FT-Raman spectroscopy, PXRD, and TG-FTIR(Table 21).

The FT-Raman spectra of class 3 are clearly similar to each other (FIG.96) but show small differences (FIG. 97). The spectra of class 3 differsignificantly from the spectrum of the amorphous starting material,class 1 (FIG. 98), and from the spectra of classes 2, 4, and 5 (FIG.72).

The PXRD patterns of class 3 (FIG. 99) confirm the crystallinity of thematerials. The patterns of the three samples are similar to each otherbut show small but significant differences (FIG. 100). The class 3pattern clearly differs from the crystalline patterns of classes 2, 4,and 5 (FIG. 75).

The TG-FTIR thermogram of sample PP415-P6 (FIG. 100) shows the loss of˜5.4 wt.-% 2PrOH from 25° C. to 250° C., most of it in a step from ˜170°C. to 190° C. Decomposition starts at temperatures T>250° C. Before theTG-FTIR experiments, the samples were dried briefly (for ˜5 min) undervacuum (10-20 mbar) to remove excess, unbound solvent. The theoretical2PrOH (b.p.=82° C.) content of a hemisolvate is 5.1 wt.-%.

The TG-FTIR thermogram of sample PP415-P12 (FIG. 101) shows the loss of˜4.9 wt.-% EtOH (with traces of water) from 25° C. to 250° C., most ofit in a step from ˜160° C. to 190° C. Decomposition starts attemperatures T>250° C. The theoretical EtOH (b.p.=78° C.) content of ahemisolvate is 4.0 wt.-%.

Thus, the samples of class 3 seem to be isostructural solvates of 2PrOH,EtOH, and probably acetone with tightly bound solvent content. Theycould correspond to stoichiometric hemisolvates. It cannot be ruled out,however, that these forms are non-stoichiometric solvates.

As the Raman spectra and PXRD patterns within this class are verysimilar to each other, the structures might be essentially identicalwith only small distortions of the unit cell dimensions or small changesof atomic positions within the unit cell due to the incorporation ofdifferent solvent molecules.

TABLE 21 Crystallization experiments resulting in solid material ofclass 3 Sample Method Solvent/Mixture Characterization Drying PP415-P6suspension 2PrOH Raman, PXRD, X equil. TG-FTIR PP415-P12 suspension  9:1EtOH/H₂O Raman, PXRD, — equil. TG-FTIR PP415-P20 slow ~2:1 acetone/H₂ORaman, PXRD — cooling

j. Drying Experiments on Samples of Class 3

One of the samples of class 3 (PP415-P6), obtained from a suspensionequilibration experiment in 2PrOH, was dried (as PP415-P25) under vacuumfor several days (2-20 mbar, r.t. to 60° C., Table 22).

The TG-FTIR thermogram of this dried class 3 material, sample PP415-P25(FIG. 102), shows the loss of ˜5.4 wt.-% 2PrOH from 50° C. to 250° C.,most of it in a step from 170° C. to 190° C., another loss of ˜1.0 wt.-%2PrOH from 290° C. to 320° C., and decomposition at temperatures T>320°C. Compared to the TG-FTIR of the original class 3 sample PP415-P6 (FIG.103), with a solvent content of ˜5.4 wt.-% 2PrOH, the solvent contentdoes not seem to have decreased significantly.

This material was dried further (as PP415-P33, Table 22) for three daysunder high vacuum and elevated temperatures (<1×10⁻³ mbar, 80° C.) withthe aim to desolvate the solvate and to obtain a non-solvated, anhydrousform of 63415.

The TG-FTIR thermogram of this further dried class 3 material, samplePP415-P33 (FIG. 103) shows the loss of ˜4.2 wt.-% 2PrOH from 50° C. to210° C., most of it in a step from 160° C. to 190° C., another loss of˜0.5 wt.-% 2PrOH from 210° C. to 290° C., and decomposition attemperatures T>290° C.

Compared to the solvent content of the samples PP415-P6 and PP415-P25,the solvent content has decreased only from ˜5.4 wt.-% to ˜4.8 wt.-%.

TABLE 22 Drying experiments on samples of class 3 Starting MaterialDried Material Solvent Drying Solvent Sample Content Conditions SampleContent Class PP415-P6 2PrOH r.t., ~3 mbar, PP415-P25 2PrOH 3 (~5.4%) ~5d; 60° C., (~5.4%) 5-10 mbar, 2 × 1 h; 40-50° C., 5-20 mbar, ~1 dPP415-P25 2PrOH 80° C., PP415-P33 2PrOH 3 (~5.4%) <1 × 10⁻³ mbar,(~4.8%) 3 d

The FT-Raman spectra of class 3 (sample PP415-P6), of the dried materialof class 3 (sample PP415-P25), and of the further dried material ofclass 3 (sample PP415-P33) are identical and show no changes (FIG. 104).

The PXRD patterns of class 3 (sample PP415-P6) and of the further driedmaterial of class 3 (sample PP415-P33) do not show any significantdifferences, while there are few small shifts and differences from thepattern of the initially dried material of class 3 (sample PP415-P25,FIG. 105). All patterns correspond to class 3.

As the drying had no major effect on the solvent content, it is notsurprising that the FT-Raman spectra and PXRD patterns of the driedmaterials do not show differences compared to the non-dried material.

Thus, class 3 is a class of isostructural solvates (2PrOH, EtOH, andprobably acetone) with very tightly bound solvent molecules that couldbe removed only partially (˜5.4 wt.-% to ˜4.8 wt.-%) by the dryingconditions applied here (up to 3 d at 1×10⁻³ mbar and 80° C.).

k. Class 4—Acetonitrile Solvate

Class 4 was obtained only from a 7:3 MeCN/H₂O solvent mixture (Table23). The experiment resulting in class 4 (PP415-P13) was repeated asPP415-P35 to prepare more material for further drying studies.

The FT-Raman spectrum (FIG. 72) and PXRD pattern (FIG. 75) of class 4(sample PP415-P13) differ significantly from the spectra and patterns ofclasses 2, 3, and 5.

The TG-FTIR thermogram of class 4 (sample PP415-P13, FIG. 106) shows theloss of ˜3.4 wt.-% MeCN (with traces of water) from 25° C. to 270° C.,most of it in a step from ˜180° C. to 210° C. Decomposition starts attemperatures T>270° C. Before the TG-FTIR experiments, the samples weredried briefly (for ˜5 min) under vacuum (10-20 mbar) to remove excess,unbound solvent. The theoretical MeCN (b.p.=81° C.) content of ahemisolvate is 3.6 wt.-%.

TABLE 23 Crystallization experiments resulting in solid material ofclass 4 Sample Method Solvent Characterization Drying PP415-P13suspension 7:3 MeCN/H₂O Raman, PXRD, X equilibration TG-FTIR PP415-P35suspension 7:3 MeCN/H₂O Raman, PXRD, X equilibration TG-FTIR

l. Drying Experiments on Class 4

The samples of class 4 obtained from suspension equilibrationexperiments in ˜7:3 MeCN/H₂O were dried under vacuum for several days orunder N₂ flow (Table 24).

TABLE 24 Drying experiments on samples of class 4 Starting MaterialDried Material Solvent Drying Solvent Sample Content Conditions SampleContent Class PP415-P13 MeCN r.t., ~3 mbar, PP415- MeCN 4 (~3.4%) ~5 d;60° C., P26 (~2.8%) 5-10 mbar, 2 × 1 h; 40-50° C., 5-20 mbar, ~1 dPP415-P35 MeCN 80° C., PP415- MeCN/H₂O^(a) 4 (~2.9%) <1 × 10⁻³ mbar, P36(~0.6%) 3 d PP415-P35 MeCN 80° C., PP415- MeCN/H₂O^(a) 4 (~2.9%) N₂flow, 3 d P37 (~0.9%) ^(a)solvent content possibly MeCN and H₂O, butdifficult to determine as amounts are small

The FT-Raman spectrum of the dried class 4 material (PP415-P26) isidentical to the spectrum of class 4 (PP415-P13, FIG. 107).

The PXRD pattern of the dried class 4 material (PP415-P26) shows onlyvery small differences from the pattern of class 4, sample PP415-P13(FIG. 108). Some peaks seem better resolved, and peak intensities haveshifted. No amorphization is observed. The pattern of PP415-P26corresponds to class 4.

The TG-FTIR thermogram of the dried class 4 material, sample PP415-P26(FIG. 109) shows the loss of ˜2.8 wt.-% MeCN from 170° C. to 250° C. anddecomposition at temperatures T>300° C. Compared to the TG-FTIR ofsample PP415-P13 (FIG. 106), the solvent content of the sample hasdecreased from 3.4 wt.-% to 2.8 wt.-%.

Thus, the sample seems to be a partially desolvated solvate. As notsufficient material remained for a second drying experiment withsubsequent characterization, experiment PP415-P13 was repeated (asPP415-P35). More material of class 4 was prepared and two dryingexperiments were carried out with this freshly prepared material:

PP415-P36: drying under vacuum (<1×10⁻³ mbar) at 80° C. for three days

PP415-P37: drying under N₂ flow at 80° C. for three days

The FT-Raman spectra of these dried class 4 samples (PP415-P36 and -P37)correspond to the spectrum of class 4 (i.e., PP415-P35, FIG. 110).

The PXRD patterns (FIG. 111) of the class 4 material (sample PP415-P35)and the dried samples of class 4 (samples PP415-P36 and PP415-P37) areidentical. The dried samples are crystalline.

The TG-FTIR thermograms of these dried samples of class 4 (FIG. 112 forPP415-P36 and FIG. 113 for PP415-P37) show only a small solvent content(MeCN and/or H₂O) of ˜0.6 wt.-% and ˜0.9 wt.-% for PP415-P36 andPP415-P37, respectively, in two steps from 25° C. to 280° C. Solventcontent is possibly MeCN and H₂O, but is difficult to determine asamounts are small. Decomposition starts at temperatures T>280° C.

Thus, most of the solvent of this solvate could be removed withoutdestroying the crystal structure. A crystalline, non-solvated form (orrather desolvated solvate) was obtained.

m. Further Characterization of the Dried and Desolvated Class 4

Drying of class 4 (MeCN solvate) resulted in a desolvated solvate withthe solvent content reduced to <1 wt.-% (TG-FTIR).

No change in the structure occurred upon desolvation (FT-Raman andPXRD). No significant loss of the crystallinity was observed.

Thus, a non-solvated, crystalline form of 63415 was obtained, the onlyone known to date.

This desolvated class 4 material was characterized further by DVS andDSC.

The DVS isotherm (FIG. 114) shows that during initial equilibration timeat 50% r.h. a mass gain of ˜0.4 wt.-% occurred. During the measurement,a gradual, reversible mass loss of ˜1.3 wt.-% occurred upon lowering therelative humidity from 50% r.h. to 0% r.h. Equilibrium was reached. Uponincreasing the relative humidity to 95% r.h., a gradual mass gain of˜0.8 wt.-% was observed (relative to the equilibration mass at 50%r.h.). Equilibrium was reached. After lowering the relative humidity to50% r.h., the final mass remained 0.1 wt.-% below the equilibratedstarting mass. The mass gain of ˜0.7 wt.-% upon increasing the relativehumidity from 50% r.h. to 85% r.h. classified the sample as slightlyhygroscopic.

The PXRD pattern of the sample after the measurement is unchangedcompared to the pattern before the measurement (FIG. 115).

The DSC thermogram of a sample of desolvated class 4 material (FIG. 116)shows no glass transition attributable to the amorphous form, whichwould have been expected at ˜150° C., but instead a sharp endothermicpeak with a maximum at T=196.1° C. (ΔH=29.31 J/g), probablycorresponding to melting, and no decomposition up to 270° C.

In addition, a DSC experiment was carried out with a ˜1:1 mixture of theamorphous material, class 1, with the desolvated class 4 material toinvestigate if the amorphous material would transform and crystallizeinto the desolvated class 4, an event expected to occur (if at all)above the glass transition temperature of the amorphous form (T_(g)≈150°C.) and below the melting of the desolvated class 4 (T_(m)≈196° C.).

The DSC thermogram of the mixture (FIG. 117) shows an endothermic eventwith a peak at T=156.7° C. (ΔH=1.47 J/g) and a second endothermic eventwith a peak at 197.0° C. (ΔH=14.1 J/g). The first event could beattributable to the amorphous material (glass transition at T_(g)≈150°C.). The second event could correspond to the melting of the desolvatedclass 4 at T_(m)≈196° C. The heat of fusion (ΔH=14.1 J/g) of the mixturecorrelates well to half of the heat of fusion (ΔH=29.3 J/g) of the puredesolvated class 4.

No exothermic event in the temperature range between the glasstransition and the melting corresponding to a possible crystallizationof the amorphous material can be observed. Thus, no transformation ofthe amorphous form into the desolvated class 4 form seemed to haveoccurred on this timescale.

In yet another DSC experiment with a ˜1:1 mixture of the amorphousmaterial, class 1, with the desolvated class 4 material, the heating wasstopped at 173° C. (in between the glass transition and the melting) toallow time for a possible crystallization.

The DSC thermogram of the mixture (FIG. 118) shows an endothermic eventwith a peak at T=161.4° C. (ΔH=0.31 J/g) and a second endothermic eventwith a peak at 201.4° C. (ΔH=11.4 J/g). As in the first experiment, theheat of fusion of the second peak did not increase; no indications for atransformation of the amorphous form into the desolvated class 4 formare visible.

The curved baseline (−50° C. to 150° C.) is most likely an artifact (dueto a bent sample holder lid).

n. Class 5—THF Solvate

Class 5 was obtained only from a 1:1 THF/H₂O solvent mixture (Table 25).

The FT-Raman spectrum (FIG. 71) and PXRD pattern (FIG. 75) of class 5differs significantly from the spectra and patterns of classes 2, 3, and4.

The TG-FTIR thermogram of class 5 (sample PP415-P14, FIG. 119) shows theloss of ˜36.1 wt.-% THF and H₂O from 25 to 200° C., most of it in a stepfrom ˜100° C. to 130° C. Before the TG-FTIR experiments, the sampleswere dried briefly (for ˜5 min) under vacuum (10-20 mbar) to removeexcess, unbound solvent. The loss of both THF and H₂O occur together inthe same temperature range. Decomposition starts at temperatures T>300°C. The theoretical THF (b.p.=66° C.) content of a trisolvate is 28.1wt.-%. Unfortunately, as the content of the two components cannot bequantified separately, the exact solvation state cannot be determined.

Details on the experiments and characterizations of samples PP415-P41and PP415-P45 are provided.

TABLE 25 Crystallization experiments resulting in solid material ofclass 5 Sample Method Solvent Characterization Drying PP415-P14suspension 1:1 THF/H₂O Raman, PXRD, X equilibration TG-FTIRPP415-P41^(b) suspension 1:1 THF/H₂O PXRD X equilibrationPP415-P45^(b,c) suspension 1:1 THF/H₂O PXRD X equilibration ^(b)startingmaterial: PP415-P40, class 2; in all other experiments in this tablePP415-P1, class 1, was used as the starting material ^(c)3-g scaleexperiment instead of 100-mg scale

o. Drying Experiments on Samples of Class 5

The sample of class 5 (PP415-P14), obtained from a suspensionequilibration experiment in ˜1:1 THF/H₂O, was dried (as PP415-P27) undervacuum for several days (2-20 mbar, r.t. to 60° C., Table 26).

TABLE 26 Drying experiments of samples of Class 5 Starting MaterialDried Material Solvent Drying Solvent Sample Content Conditions SampleContent Class PP415- THF & H₂O r.t., ~3 mbar, PP415- — 1 (+5)^(a) P14(~36 wt.-%) ~5 d; 60° C., P27 (~0.3 wt.-%) 5-10 mbar, 2 × 1 h; 40-50°C., 5-20 mbar, ~1 d ^(a)mainly amorphous, only few broad peaks with lowS/N ratio

The FT-Raman spectrum of the dried material (PP415-P27) is differentfrom the spectrum of class 5 (PP415-P14, FIG. 120) and, with itsbroadened peaks, resembles more the spectrum of class 1, the amorphousstarting material, PP415-P1.

The PXRD pattern of the dried class 5 material (PP415-P27) shows onlysome broad, low intensity peaks with a low S/N ratio, indicating thepoor crystallinity of the sample (FIG. 121). Some of the peaks couldcorrespond to class 5, while others, i.e., at 7.35°2θ, are new orshifted.

The TG-FTIR thermogram of the dried class 5 material (FIG. 122) shows amass loss of ˜0.3 wt.-% from 25° C. to 290° C. and decomposition attemperatures T>290° C. The sample is anhydrous.

Thus, by drying under vacuum, the material has lost its solvent contentand also much of its crystallinity.

8. Experiments to Prepare the Amorphous Form

Experiments with the aim to prepare the amorphous form, class 1, werecarried out using class 2 material (PP415-P40, Table 8) as the startingmaterial. Several strategies and methods were attempted:

-   -   Transformation of class 2 into class 5, followed by drying of        class 5 to obtain the amorphous form, class 1.    -   Preparation of the amorphous form, class 1, directly from class        2, if possible using ICH class 3 solvents.        Mainly amorphous material was prepared starting from class 2        material in a two-step process via class 5 on a 100-mg and 3-g        scale.

Further experiments were carried out with the aim to simplify theprocedure to a one-step process, to avoid the ICH class 2 solvent THF,and to obtain fully amorphous material. The most promising method wasfound to be the precipitation from an acetone solution in a cold waterbath. This direct method gives much better results than the two-stepmethod via class 5.

a. Preparation of the Amorphous Form Via Class 5

Crystallization experiments using class 2, PP415-P40, as the startingmaterial were carried out with the aim to transform this heptane solvateinto class 5 (likely THF solvate), followed by drying of class 5 toobtain the amorphous material (Table 27).

Class 5 is thought to be a good intermediate step, as it is easier todesolvate and amorphize than classes 2 or 3.

TABLE 27 Summary of experiments aimed at preparation of amorphous form,class 1, via class 5 material Step Sample Method Conditions Results 1PP415-P41 suspension 1:1 THF/H₂O, 24° C., class 5 equil. 3 d ″ PP415-P45suspension 1:1 THF/H₂O, r.t. 1 d class 5 equil. 2 PP415-P44a drying 100mbar, 80° C., 2 d class 1^(a); 0.9 wt.-% THF ″ PP415-P46a drying 100mbar, 80° C., 4 d class 1^(a); 0.4 wt.-% H₂O ^(a)mainly amorphous, onlyfew broad peaks with low S/N ratio

b. Step 1: Transformation of Class 2 into Class 5

Transformation of the heptane solvate, class 2, into the THF solvate,class 5, was successfully carried out by suspending the PP415-P40(heptane solvate) material in a (1:1) THF/H₂O mixture and equilibratingthe suspension at r.t. (PP415-P41, 100 mg-scale). The resulting solidmaterial corresponds to the THF solvate, class 5 (FIG. 123).

A first scale-up experiment from the mg-scale to the g-scale (×30, i.e.,3-g scale) was carried out analogous to PP415-P41: the class 2 heptanesolvate starting material (PP415-P40) was equilibrated in THF/H₂O (1:1)for one day and successfully transformed into class 5, the THF solvate(PP415-P45, FIG. 124).

c. Step 2: Amorphization of Class 5 Material by Drying

The class 5 material (THF solvate) was dried at elevated temperature(80° C.) under vacuum (˜100 mbar) taking into account the conditionsthat can be used at the API MFG site.

After drying the material of the 100-mg scale experiment, PP415-P41, forone day at 80° C. and 100 mbar it transformed into mainly amorphousmaterial (PP415-P44, FIG. 125). The PXRD pattern shows only some broadpeaks with low intensity. After additional drying (80° C., 100 mbar)overnight, the intensity of these broad peaks is further reduced(PP415-P44a). The TG-FTIR of this material shows the loss of ˜0.9 wt.-%THF (with traces of water) gradually from 25° C. to 280° C. anddecomposition at temperatures T>300° C. (FIG. 126).

The material of the 3-g scale experiment, PP415-P45, was also dried at80° C. and 100 mbar (as PP415-P46). It transformed overnight into mainlyamorphous material with only some broad peaks with low intensity (FIG.127). After a total of four days of drying (80° C., 100 mbar), thesebroad peaks are still present (-P46a, FIG. 128). The TG-FTIR of thismaterial shows no THF content, but the loss of ˜0.4 wt.-% watergradually from 25° C. to 250° C. and decomposition at temperaturesT>250° C. (FIG. 129).

d. Obtaining the Amorphous Form Directly

The preparation of the amorphous form starting from class 2 material inthe two-step process via class 5 was largely, but not fully, successful.Thus, further experiments were carried out with the aim to simplify theprocedure to a one-step process, to avoid the use of ICH class 2 solventTHF, and to obtain fully amorphous material (Table 28).

The amorphous form, class 1, was prepared directly from the class 2material in an evaporation experiment of a class 2 solution in THF underN₂ flow (PP415-P42, FIG. 129).

In an attempt to simulate an incompletely dried heptane/hexane solvatewith a significant amount of remaining solvent, an evaporation of aclass 2 solution in an 8:2 THF/hexane solution was carried out (hexanewas used instead of heptane in order to have similar boiling points inthe solvent mixture). However, the resulting solid corresponds to class2, the class of the isostructural solvates, not to class 5 (PP415-P43,FIG. 130).

In order to avoid the ICH class 2 solvent THF, evaporation experimentswere carried out in ICH class 3 solvents.

Evaporation of a class 2 solution in EtOAc under N₂ flow resulted incrystalline material with a PXRD pattern corresponding to class 2(PP415-P47, FIG. 130). The TG-FTIR (FIG. 80) shows the class 2-typicaltwo-step mass loss (total of ˜7.9 wt.-% EtOAc) at temperatures up to240° C., indicating very tightly bound solvent molecules.

Evaporation in ethyl formate also gave crystalline class 2 material andnot the amorphous form (PP415-P48, FIG. 131). The TG-FTIR (FIG. 78)shows the mass loss of ˜3.5 wt.-% ethyl formate, at first gradually andthen in a clear step between 180° C. and 200° C. There might be furtherloss of ethyl formate concomitant with the decomposition at T>240° C.

However, class 2 material was successfully transformed into theamorphous form, class 1, by adding an acetone solution to a cold (5° C.)water bath (PP415-P49, FIG. 132).

This direct method for the preparation of the amorphous form givesbetter results and is a more promising route than the two-step process.

TABLE 28 Summary experiments aimed at obtaining the amorphous formdirectly from class 2 starting material Sample Method Solvent ConditionResult PP415-P42 evaporation THF N₂ flow, 1 d class 1 PP415-P43evaporation 8:2 THF/hexane N₂ flow, 1 d class 2 PP415-P47 evaporationEtOAc N₂ flow, 1 d class 2 PP415-P48 evaporation ethyl formate N₂ flow,1 d class 2 PP415-P49 precipitation acetone H₂O bath class 1 at 5° C.

9. Instrumental—Typical Measurement Conditions

FT-Raman Spectroscopy: Bruker RFS100 with OPUS 6.5 software; Nd:YAG1064-nm excitation, Ge detector, 3500-100 cm⁻¹ range; typicalmeasurement conditions: 100-300 mW nominal laser power, 64-128 scans, 2cm⁻¹ resolution.

PXRD: Stoe Stadi P; Mythen1K Detector; Cu-Kα radiation; standardmeasurement conditions: transmission; 40 kV and 40 mA tube power; curvedGe monochromator; 0.02°2θ step size, 12 s or 60 s step time, 1.5-50.5°2θor 1.0-55°2θ scanning range; detector mode: step scan; 1°2θ or 6°2θdetector step; standard sample preparation: 10 to 20 mg sample wasplaced between two acetate foils; sample holder: Stoe transmissionsample holder; the sample was rotated during the measurement.

TG-FTIR: Netzsch Thermo-Microbalance TG 209 with Bruker FT-IRSpectrometer Vector 22; aluminum crucible (with microhole), N₂atmosphere, 10 K/min heating rate, 25-250° C. or 25-350° C. range.

DSC: Perkin Elmer DSC 7; gold crucibles (closed or with microhole),sample filled in an N₂ environment, 10 K/min heating rate, −50 to 250°C. or 350° C. range, at times quench cooling (at −200 K/min) to −50° C.between scans.

DVS: Projekt Messtechnik Sorptions Prüfsystem SPS 11-100n or SurfaceMeasurement Systems DVS-1. The sample was placed on an aluminum orplatinum holder on top of a microbalance and allowed to equilibrate for2 h at 50% r.h. before starting the pre-defined humidity program:

(1) 50→0% r.h. (5%/h); 5 h at 0% r.h.

(2) 0→95% r.h. (5%/h); 5 h at 95% r.h.

(3) 95→50% r.h. (5%/h); 2 h at 50% r.h.

The hygroscopicity was classified based on the mass gain at 85% r.h.relative to the initial mass as follows: deliquescent (sufficient wateradsorbed to form a liquid), very hygroscopic (mass increase of ≧15%),hygroscopic (mass increase <15% but ≧2%), slightly hygroscopic (massincrease <2% but ≧0.2%), or non-hygroscopic (mass increase <0.2%).

Solvents: For all experiments, Fluka, Merck, or ABCR analytical gradesolvents were used.

Approximate Solubility Determination: Approximate solubilities weredetermined by a stepwise dilution of a suspension of about 10 mg ofsubstance in 0.05 mL of solvent. If the substance was not dissolved byaddition of a total of >10 mL solvent, the solubility is indicated as <1mg/mL. Due to the experimental error inherent in this method, thesolubility values are intended to be regarded as rough estimates and areto be used solely for the design of crystallization experiments.

Chemical Stability Determination: Four samples of 1.0 mg of the PP415-P1material in 1.0 mL of the respective solvent were prepared. Theresulting suspensions/solutions were equilibrated in atemperature-controlled Eppendorf Thermomixer Comfort shaker for 7 d, 2d, 24 h, and 6 h at 25° C. at a shaking rate of 500 rpm. If necessary,the solid phase was separated by filter centrifugation (0.5-μm PVDFmembrane). The filtrates were diluted in the diluent (0.1% formic acidin MeCN) to concentrations ≦0.2 mg/mL (unknown and likely lower forsuspensions) and examined using the HPLC method given in Table 29. Asreference, the PP415-P1 material was diluted in the diluent to aconcentration of 0.25 mg/mL and added to the beginning and end of theHPLC sequence.

HPLC Results

TABLE 29 HPLC method used for chemical stability determinations ColumnZorbax Eclipse XDB-C18, 3 × 150 mm, 5 μm (CC19) Eluent A H₂O + 0.1%formic acid Eluent B MeCN + 0.1% formic acid Gradient  0 min 50% A 50% B10.0 min 10% A 90% B 15.0 min  0% A 100% B  15.1 min 50% A 50% B 20.0min 50% A 50% B Flow 0.75 mL/min Injection Volume 10 μL Wavelength 254nm, 242 nm, 210 nm Acquisition time 20 min Run time 20 min Column 25° C.temperature Retention time 8.9-9.0 min

10. Abbreviations

Methods:

-   -   AUC area under the curve analysis    -   DSC differential scanning calorimetry    -   DVS dynamic vapor sorption    -   FT Raman Fourier-transform Raman spectroscopy    -   ¹H-NMR proton nuclear magnetic resonance spectroscopy    -   HPLC high-performance liquid chromatography    -   PXRD powder X-ray diffraction    -   TG-FTIR thermogravimetry coupled to Fourier transform infrared        spectroscopy

Chemicals:

-   -   1BuOH 1-butanol    -   CTAB cetyl trimethylammonium bromide    -   DCM dichloromethane    -   DEE diethyl ether    -   DMF N,N-dimethylformamide    -   EtOAc ethyl acetate    -   EtOH ethanol    -   IPE isopropyl ether    -   MeCN acetonitrile    -   MEK methyl ethyl ketone    -   MeOH methanol    -   PEG propylene glycol    -   PTFE polytetrafluoroethylene, Teflon    -   2PrOH 2-propanol, isopropanol    -   SDS sodium dodecyl sulfate    -   TBME tert-butyl methyl ether    -   TEA triethylamine    -   THF tetrahydrofuran    -   Tween 80 polyoxyethylene (80) sorbitan monooleate or polysorbate        80

Genes, Proteins, and Biological Parameters:

-   -   AIM antioxidant inflammation modulator    -   Akr1c1 aldo-keto reductase family 1, member c1    -   ALP alkaline phosphatase    -   ALT alanine transaminase    -   ARE antioxidant response element    -   AST aspartate transaminase    -   AUC area under the curve    -   BAL bronchoalveolar lavage    -   BALF bronchoalveolar lavage fluid    -   Bil bilirubin    -   BUN blood urea nitrogen    -   COPD chronic obstructive pulmonary disease    -   COX-2 cyclooxygenase-2    -   Cr creatine    -   CYP450 cytochrome P450    -   Eh-1 epoxide hydrolase 1    -   G6PD glucose-6 phosphate dehydrogenase    -   Gclc glutamate-cysteine ligase, catalytic subunit    -   Gclm glutamate-cysteine ligase, modifier subunit    -   Ggtl gamma-glutamyltransferase    -   Glrx glutaredoxin-1    -   Glu glucose    -   GOT glutamic-oxaloacetic transaminase    -   GPT1 glutamic-pyruvate transaminase    -   Gpx3 glutathione peroxidase 3    -   GSH glutathione    -   GSR glutathione reductase    -   GSs glutathione synthetase    -   GST glutathione S-transferase    -   GSTa1 glutathione S-transferase alpha 1    -   GSTp1 glutathione S-transferase pi 1    -   Gy Gray    -   H6PD hexose-6-phosphate dehydrogenase    -   hERG human ether a-go-go-related gene    -   HMOX1 heme oxygenase (decycling) 1    -   HO-1 heme oxygenase    -   IFNγ interferon-gamma    -   IL interleukin    -   iNOS inducible nitric oxide synthase    -   IκBα nuclear factor of kappa light polypeptide gene enhancer in    -   B-cells inhibitor, alpha    -   KC mouse IL-8 related protein    -   Keap1 Kelch-like ECH associated protein-1    -   LPS lipopolysaccharide    -   ME1 malic enzyme 1    -   MPCE micronucleated polychromatic erythrocytes    -   Mrp metG-related protein    -   Mrps multidrug resistance-related proteins    -   NADPH nicotinamide adenine dinucleotide phosphate, reduced    -   NFκB nuclear factor of kappa-light-chain-enhancer of activated B        cells    -   NO nitric oxide    -   NQO1 NAD(P)H quinone oxidoreductase 1    -   Nrf2 nuclear factor (erythroid-derived)-like 2    -   p-IκBα phosphorylated IκBα    -   PBMC peripheral blood mononuclear cell    -   PCE polychromatic erythrocytes    -   PGD phosphogluconate dehydrogenase    -   PMN polymorphonuclear    -   RANTES regulated and normal T cell expressed and secreted    -   SOD1 superoxide dismutase 1    -   SRXN1 sulfuredoxin-1    -   TG total glycerides    -   TKT transketolase    -   TNFα tumor necrosis factor alpha    -   Txn thioredoxin    -   TXNRD 1 thioredoxin reductase 1    -   xCT solute carrier family7, member 11

Misc:

-   -   API active pharmaceutical ingredient    -   aq. aqueous    -   b.p. boiling point    -   cryst. crystalline    -   decomp. decomposition    -   d day(s)    -   eq. equivalent    -   equil. equilibration    -   evap. evaporation    -   h hour(s)    -   mat. material    -   min minute(s)    -   m.p. melting point    -   MS molecular sieves    -   part. partially    -   precip. precipitation    -   r.h. relative humidity    -   rpm revolutions per minute    -   r.t. room temperature (˜25° C.)    -   S/N signal-to-noise (ratio)    -   solv. solvent    -   susp. suspension    -   T temperature        -   T_(g) glass transition temperature    -   theo. theoretical    -   vis. obs. visual observation    -   w week(s)    -   wt.-% weight percent

K. Further Tables

TABLE 30 List of Samples and Performed Experiments Sample ExperimentalDescription Test Methods Result/Remarks PP415-P1 received ~5 g of 63415,batch # 0141-66-1, on Mar. FT-Raman: FT-Raman: used as reference for P125, 2011; MW = 554.7 g/mol, C₃₃H₄₄F₂N₂O₃ PP415P1.0 PXRD: amorphous, nocrystalline PP415P1.1 peak pattern PXRD: 117a TG-FTIR: loss of ~0.9wt.-% TG-FTIR: a4285 (~0.1 eq.) EtOH with traces of H₂O ¹H-NMR: from 25°C. to 200° C., decomposition Mar30-2011- at T >290° C. ktr/30 ¹H-NMR:agrees with structure, ~0.08 DSC: d_9840 eq. EtOH DVS: #0305_02 DSC:1^(st) scan: Tg = 152.7° C. (ΔCp = post-DVS Raman: 0.72 J/g° C.); 2ndscan: Tg = 149.7° C. PP415P1_aDVS (ΔCp = 0.45 J/g° C.) post-DVS PXRD:DVS: slightly hygroscopic; Δm = 179a +0.4% (50%→85% r.h.); total massgain of 2.1 wt.-% from 0% r.h. to 95% r.h. post-DVS Raman and PXRD:unchanged PP415-P2 stored material of PP415-P1 at 25° C. open over aPXRD: PXRD: all amorphous, correspond to saturated solution of NH₄NO₃(i.e., at ~62% r.h.); 132a (-P2a) P1 examined samples after 1 w(PP415-P2a), 2 w (PP415-P2b), and 4 w 191a (-P2b) (PP415-P2c). 262a(-P2c) PP415-P3 stored material of PP415-P1 at 40° C. open over a PXRD:PXRD: all amorphous, correspond to saturated solution of NaCl (i.e., at~75% r.h.); examined 133a (-P3a) P1 samples after 1 w (PP415-P3a), 2 w(PP415-P3b), and 4 192a (-P3b) w (PP415-P3c). 263a (-P3c) PP415-P4stored material of PP415-P1 at 60° C. in a closed PXRD: PXRD: allamorphous, correspond to container; examined samples after 1 w(PP415-P4a), 2 w 134a (-P4a) P1 (PP415-P4b), and 4 w (PP415-P4c). 193a(-P4b) 264a (-P4c) PP415-P5 stored material of PP415-P1 at 80° C. in aclosed PXRD: PXRD: all amorphous, correspond to container; examinedsamples after 1 w (PP415-P5a), 2 w 135a (-P5a) P1 (PP415-P5b), and 4 w(PP415-P5c). 194a (-P5b) 265a (-P5c) PP415-P6 suspended 97.7 mg ofPP415-P1 in 0.4 mL of 2PrOH; FT-Raman: Raman: corresponds to class 3obtained white suspension; equilibrated suspension at 22° C. PP415P6.0PXRD: corresponds to class 3 shaking at 400 rpm; added stepwise a totalof 0.5 mL PXRD: 225a TG-FTIR: loss of ~5.4 wt.-% 2PrOH of the solventover the next couple of days; after 15 d TG-FTIR: a4323 from 25° C. to250° C., most of it in a recovered solid material by filtercentrifugation (0.20-μm step from ~170° C. to 190° C.; PTFE membrane);examined material by FT-Raman decomposition starts at T>250° C.spectroscopy and PXRD; dried material for 5 min under vacuum (10-20mbar); examined material by TG-FTIR. PP415-P7 suspended 104.3 mg ofPP415-P1 in 0.6 mL of FT-Raman: Raman: corresponds to class 2 1:2EtOAc/heptane; obtained white suspension; PP415P7.0 PXRD: corresponds toclass 2 equilibrated suspension at 22° C. shaking at 400 rpm; PXRD: 227aTG-FTIR: loss of ~7.5 wt.-% EtOAc added stepwise a total of 0.2 mL ofthe solvent mixture TG-FTIR: a4338 and heptane in two steps from overthe next couple of days; after 15 d recovered solid ~100° C. to 290° C.;decomposition material by filter centrifugation (0.20-μm PTFE starts atT >290° C. membrane); examined material by FT-Raman spectroscopy andPXRD; dried material for 5 min under vacuum (10-20 mbar); examinedmaterial by TG-FTIR. PP415-P8 suspended 102.0 mg of PP415-P1 in 0.4 mLof 1:2 FT-Raman: Raman: corresponds to class 2 acetone/hexane; obtainedwhite suspension; equilibrated PP415P8.0 PXRD: corresponds to class 2suspension at 22° C. shaking at 400 rpm; added stepwise PXRD: 228a atotal of 0.2 mL of the solvent mixture over the next couple of days;after 15 d recovered solid material by filter centrifugation (0.20-μmPTFE membrane); examined material by FT-Raman spectroscopy and PXRD.PP415-P9 suspended 102.6 mg of PP415-P1 in 0.4 mL of 1:3 FT-Raman:Raman: corresponds to class 2, toluene/diethyl ether; obtained whitesuspension; PP415P9.0 contains solvent signals equilibrated suspensionat 22° C. shaking at 400 rpm; added stepwise a total of 0.2 mL of thesolvent mixture over the next couple of days; after 15 d recovered solidmaterial by filter centrifugation (0.20-μm PTFE membrane); examinedmaterial by FT-Raman spectroscopy. PP415-P10 suspended 102.5 mg ofPP415-P1 in 0.2 mL of FT-Raman: Raman: corresponds to class 2 1:3MeOH/TBME; obtained clear solution; equilibrated PP415P10.0 PXRD:corresponds to class 2 solution at 22° C. shaking at 400 rpm; after 1 dobserved PXRD: 229a thick suspension; added 0.2 mL of the solventmixture; continued equilibration of suspension at 22° C. shaking at 400rpm; after a total of 15 d recovered solid material by filtercentrifugation (0.20-μm PTFE membrane); examined material by FT-Ramanspectroscopy and PXRD. PP415-P11 suspended 97.1 mg of PP415-P1 in 0.4 mLof 1:2 FT-Raman: Raman: corresponds to class 2, MEK/cyclohexane;obtained white suspension; PP415P11.0 contains solvent signalsequilibrated suspension at 22° C. shaking at 400 rpm; after 15 drecovered solid material by filter centrifugation (0.20-μm PTFEmembrane); examined material by FT-Raman spectroscopy. PP415-P12suspended 98.6 mg of PP415-P1 in 0.2 mL of 9:1 FT-Raman: Raman:corresponds to class 3 EtOH/H₂O; obtained white suspension; equilibratedPP415P12.0 PXRD: corresponds to class 3 suspension at 22° C. shaking at400 rpm; added stepwise PXRD: 230a TG-FTIR: loss of ~4.9 wt.-% EtOH atotal of 0.2 mL of the solvent mixture over the next TG-FTIR: a4324(with traces of water) from 25° C. to couple of days; after 15 drecovered solid material by 250° C., most of it in a step from filtercentrifugation (0.20-μm PTFE membrane); ~160° C. to 190° C.;decomposition examined material by FT-Raman spectroscopy and starts atT>250° C. PXRD; dried material for 5 min under vacuum (10-20 mbar);examined material by TG-FTIR. PP415-P13 suspended 95.9 mg of PP415-P1 in0.2 mL of 7.3 FT-Raman: Raman: corresponds to class 4, MeCN/H₂O;obtained two clear, separated phases; PP415P13.0 contains solventsignals equilibrated solution at 22° C. shaking at 400 rpm; after PXRD:231a PXRD: corresponds to class 4 1 d observed thick suspension; added0.2 mL of the TG-FTIR: a4321 TG-FTIR: loss of ~3.4 wt.-% MeCN solventmixture; continued equilibration of suspension (with traces of water)from 25° C. to at 22° C. shaking at 400 rpm; after a total of 15 d 270°C., most of it in a step from recovered solid material by filtercentrifugation (0.20-μm ~180° C. to 210° C.; decomposition PTFEmembrane); examined material by FT-Raman starts at T>270° C.spectroscopy and PXRD; dried material for 5 min under vacuum (10-20mbar); examined material by TG-FTIR. PP415-P14 suspended 95.8 mg ofPP415-P1 in 0.2 mL of FT-Raman: Raman: corresponds to class 5, 9:1THF/H₂O; obtained two clear, separated phases; PP415P14.0 containssolvent signals equilibrated solution at 22° C. shaking at 400 rpm;after PXRD: 232a PXRD: corresponds to class 5 1 d observed one clearphase; added 0.2 mL of H₂O; TG-FTIR: a4322 TG-FTIR: loss of ~36.1 wt.-%THF observed white precipitate; equilibrated suspension at 22° C. andH₂O from 25° C. to 200° C., most shaking at 400 rpm; after a total of 15d recovered of it in a step from ~100° C. to 130° C.; solid material byfilter centrifugation (0.20-μm PTFE decomposition starts at T >300° C.membrane); examined material by FT-Raman spectroscopy and PXRD; driedmaterial for 5 min under vacuum (10-20 mbar); examined material byTG-FTIR. PP415-P15 dissolved 100.6 mg of PP415-P1 in 0.2 mL of 1:2FT-Raman: Raman: corresponds to class 2, DCM/IPE; obtained clearsolution; observed PP415P15.0 contains solvent signals precipitation ofwhite solid in <1 min; added 0.2 mL of PXRD: 137a PXRD: corresponds toclass 2 solvent mixture; covered vial with single-layer tissue and letsolvent evaporate under ambient conditions; obtained wet, white solidmaterial after several hours; examined solid by FT-Raman spectroscopyand PXRD. PP415-P16 dissolved 100.3 mg of PP415-P1 in 0.2 mL of 1:2FT-Raman: FT-Raman: corresponds to class 1, MeOH/toluene; obtained clearsolution; covered vial PP415P16.0 contains toluene solvent peaks withsingle-layer tissue and let solvent evaporate under ambient conditions;obtained glassy material after several days; examined material byFT-Raman spectroscopy. PP415-P17 dissolved 101.0 mg of PP415-P1 in 0.3mL of 1:3 FT-Raman: Raman: corresponds to class 2, EtOAc/heptane;obtained clear solution; observed PP415P17.0 contains solvent signalsprecipitation of white solid in <1 min; added 0.2 mL of PXRD: 138a PXRD:corresponds to class 2 solvent mixture; covered vial with single-layertissue and let solvent evaporate under ambient conditions; obtained wet,white solid material after several hours; examined solid by FT-Ramanspectroscopy and PXRD. PP415-P18 dried material of PP415-P15 undervacuum (2-20 mbar) FT-Raman: Raman: corresponds to class 2 at r.t. for~2 h; examined dry, white solid by FT-Raman PP415P18.0 PXRD: correspondsto class 2 spectroscopy, PXRD, and TG-FTIR. PXRD: 149a TG-FTIR: loss of~7.0 wt.-% IPE in TG-FTIR: a4301 two steps from ~140° C. to ~250° C.;decomposition at T >250° C. PP415-P19 dried material of PP415-P17 undervacuum (2-20 mbar) FT-Raman: Raman: corresponds to class 2 at r.t. for~2 h; examined dry, white solid by FT-Raman PP415P19.0 PXRD: correspondsto class 2 spectroscopy, PXRD, and TG-FTIR. PXRD: 150a TG-FTIR: loss of~7.6 wt.-% heptane TG-FTIR: a4302 in two steps from ~140° C. to ~270°C.; decomposition at T >270° C. PP415-P20 suspended 98.8 mg of PP415-P1in 2.0 mL of H₂O; FT-Raman: Raman: corresponds to class 3, heatedsuspension to 50° C.; added slowly and stepwise PP415P20.0 containssolvent signals 4.0 mL of acetone; obtained clear solution; heated PXRD:226a PXRD: corresponds to class 3 solution to 55° C. and held at 55° C.for 30 min; slowly cooled in 4 h 10 min to 5° C. (at ~0.2 K/min);recovered solid by vacuum filtration (P4 pore size); examined solid byFT-Raman spectroscopy and PXRD. PP415-P21 suspended 100.9 mg of PP415-P1in 2.0 mL of FT-Raman: Raman: corresponds to class 2 cyclohexane; heatedsuspension to 70° C.; added slowly PP415P21.0 PXRD: corresponds to class2 and stepwise 0.5 mL of cyclohexane and 0.5 mL of PXRD: 218a TG-FTIR:loss of ~5.8 wt.-% EtOH; thin suspension became thicker due toadditional TG-FTIR: a4326 cyclohexane in two steps from ~140° C.precipitation over course of solvent addition; heated to ~250° C.;decomposition at T >250° C. suspension to 75° C. and held at 75° C. for30 min; slowly cooled in 5 h to 5° C. (at ~0.23 K/min); recovered solidby vacuum filtration (P4 pore size); examined solid by FT-Ramanspectroscopy and PXRD; dried material for 5 min under vacuum (10-20mbar); examined material by TG-FTIR. PP415-P22 suspended 151.1 mg ofPP415-P1 in 1.5 mL of toluene; FT-Raman: Raman: corresponds to class 1,heated suspension to 70° C.; obtained clear solution; PP415P22.0contains solvent signals added 0.5 mL of MeCN; heated solution to 75° C.and held at 75° C. for 30 min; slowly cooled in 5 h to 5° C. (at ~0.23K/min); observed clear solution and no precipitation; stirred clearsolution at 5° C. for 2 d; observed no precipitation; evaporated solventunder N₂ flow at r.t.; obtained glassy substance; examined it byFT-Raman spectroscopy. PP415-P23 suspended 150.6 mg of PP415-P1 in 1.5mL of dioxane; FT-Raman: Raman: corresponds to class 1, heatedsuspension to 70° C.; obtained clear solution; PP415P23.0 containssolvent signals added 0.5 mL of EtOAc; heated solution to 75° C. andheld at 75° C. for 30 min; slowly cooled in 5 h to 5° C. (at ~0.23K/min); observed clear solution and no precipitation; stirred clearsolution at 5° C. for 2 d; observed no precipitation; evaporated solventunder N₂ flow at r.t.; obtained glassy substance; examined it byFT-Raman spectroscopy. PP415-P24 suspended 99.4 mg of PP415-P1 in 0.3 mLof 1BuOH; FT-Raman: Raman: corresponds to class 2, heated suspension to70° C.; obtained clear solution; PP415P24.0 contains solvent signalsobserved shortly thereafter precipitation of white solid; PXRD: 219aPXRD: corresponds to class 2 added 0.5 mL 1BuOH; still suspension;heated TG-FTIR: a4325 TG-FTIR: loss of ~16.6 wt.-% 1BuOH suspension to75° C. and held at 75° C. for 30 min; slowly in a step from ~50° C. to~160° C., cooled in 5 h to 5° C. (at ~0.23 K/min); recovered solidfurther loss of 1BuOH (6.6 wt.-%) in a by vacuum filtration (P4 poresize); examined solid by second step from 160° C. to 230° C.; FT-Ramanspectroscopy and PXRD; dried material for 5 min decomposition at T >230°C. under vacuum (10-20 mbar); examined material by TG-FTIR. PP415-P25dried material of PP415-P25 under vacuum: at 60° C. FT-Raman: Raman:corresponds to class 3 and ~5 mbar for ~1 h; at r.t. and ~3 mbar for 4.5d; at PP415P25.0 PXRD: corresponds to class 3 60° C. and ~10 mbar for 1h; at 40-50° C. and 5-20 mbar PXRD: 258a TG-FTIR: loss of ~5.4 wt.-%2PrOH for ~20 h, examined solid by FT-Raman spectroscopy, TG-FTIR: a4337from 50° C. to 250° C., most of it in a PXRD, and TG-FTIR. step from170° C. to 190° C., another loss of ~1.0 wt.-% 2PrOH from 290° C. to320° C.; decomposition at T >320° C. PP415-P26 dried material ofPP415-P13 under vacuum: at 60° C. FT-Raman: Raman: corresponds to class4 and ~5 mbar for ~1 h; at r.t. and ~3 mbar for 4.5 d; at 60° C.PP415P26.0 PXRD: corresponds to class 4 and ~10 mbar for 1 h; at 40-50°C. and 5-20 mbar for PXRD: 259a TG-FTIR: loss of ~2.8 wt.-% MeCN ~20 h,examined solid by FT-Raman spectroscopy, TG-FTIR: a4335 from 170° C. to250° C.; decomposition PXRD, and TG-FTIR. at T >300° C. PP415-P27 driedmaterial of PP415-P14 under vacuum: at 60° C. FT-Raman: Raman: seems tocorresponds to a and ~5 mbar for ~1 h; at r.t. and ~3 mbar for 4.5 d; at60° C. PP415P27.0 mixture of class 1 and class 5 and ~10 mbar for 1 h;at 40-50° C. and 5-20 mbar for PXRD: 260a PXRD: sample is only partially~20 h, examined solid by FT-Raman spectroscopy, TG-FTIR: a4336crystalline; the few, broad peaks PXRD, and TG-FTIR. correspond to class5; thus corresponds to a mixture of the amorphous class 1 and class 5TG-FTIR: loss of ~0.3 wt.-% from 25° C. to 290° C.; decomposition atT >290° C. PP415-P28 dried material of PP415-P21 under vacuum: at 60° C.FT-Raman: Raman: corresponds to class 2 and ~5 mbar for ~1 h; at r.t.and ~3 mbar for 4.5 d; at 60° C. PP415P28.0 PXRD: corresponds to class2, sample and ~10 mbar for 1 h; at 40-50° C. and 5-20 mbar for PXRD:261a less crystalline, as indicated by ~20 h, examined solid by FT-Ramanspectroscopy, TG-FTIR: a4334 broader peaks PXRD, and TG-FTIR. TG-FTIR:loss of ~3.0 wt.-% cyclohexane in two steps from ~140° C. to ~250° C.;decomposition at T >250° C. PP415-P29 suspended 132.2 mg of PP415-P1 in0.8 mL of 1:2 FT-Raman: Raman: corresponds to class 2 EtOAc/TEA;observed change in appearance of solid PP415P29.0 PXRD: corresponds toclass 2 phase; agitated and sonicated; equilibrated suspension at PXRD:282a TG-FTIR: loss of ~5.1 wt.-% EtOAc 24° C. shaking at 500 rpm; after4 d recovered solid TG-FTIR: a4346 and TEA from ~50° C. to ~220° C.,material by filter centrifugation (0.20-μm PTFE most of it in a stepfrom 180° C. to 210° C.; membrane); examined material by FT-Ramandecomposition at T >220° C. spectroscopy and PXRD; dried material for 5min under vacuum (10-20 mbar); examined material by TG-FTIR. PP415-P30dried material of PP415-P7 under vacuum at 50-70° C. FT-Raman: Raman:corresponds to class 2 and 1-10 mbar for 3 days; examined solid byFT-Raman PP415P30.0 PXRD: corresponds to class 2 spectroscopy, PXRD, andTG-FTIR. PXRD: 290a TG-FTIR: loss of ~2.1 wt.-% heptane TG-FTIR: a4347(and some EtOAc) in two steps from ~50° C. to ~250° C.; decomposition atT >250° C. PP415-P31 suspended 137.6 mg of PP415-P1 in 2 mL of 9:1FT-Raman: Raman: corresponds to class 1 H₂O/PEG 400; obtained whitesuspension; equilibrated PP415P31.0 PXRD: amorphous, corresponds tosuspension at 24° C. shaking at 400 rpm; after 5 d PXRD: 320a class 1recovered solid material by vacuum filtration; washed solid three timeswith small amount of H₂O; examined material by FT-Raman spectroscopy andPXRD. PP415-P32 dried material of PP415-P19 under vacuum at 80° C. andFT-Raman: Raman: corresponds to class 2 <1 × 10⁻³ mbar; after 1 dexamined material by TG-FTIR PP415P32.0 PXRD: corresponds to class 2,less (a4362); continued drying; after a total of 3 d examined PXRD: 331acrystalline material by TG-FTIR (a4365), FT-Raman spectroscopy, (P32A)TG-FTIR P32: loss of 2.8 wt.-% and PXRD as P32A. TG-FTIR: heptane(25-250° C.), most of it in a a4362(P32) step from 170° C. to 200° C.;a4365 (P32A) decomposition at T >250° C. TG-FTIR P32A: loss of 2.2 wt.-%heptane (25-250° C.), most of it in a step from 170° C. to 200° C.;decomposition at T >250° C. PP415-P33 dried material of PP415-P19 undervacuum at 80° C. and FT-Raman: Raman: corresponds to class 3 <1 × 10−3mbar; after 3 d examined material by FT- PP415P33.0 PXRD: corresponds toclass 3 Raman spectroscopy, PXRD and TG-FTIR. PXRD: 332a TG-FTIR: lossof ~4.2 wt.-% 2PrOH TG-FTIR: a4366 (50-210° C.), most of it in a stepfrom 160° C. to 190° C., another loss of ~0.5 wt.- % 2PrOH (210° C. to290° C.); decomposition at T >290° C. PP415-P34 dried material ofPP415-P19 under vacuum at 80° C. and FT-Raman: Raman: corresponds toclass 2 <1 × 10−3 mbar; after 3 d examined material by FT- PP415P34.0PXRD: corresponds to class 2, less Raman spectroscopy, PXRD and TG-FTIR.PXRD: 333a crystalline TG-FTIR: a4367 TG-FTIR: loss of ~2.3 wt.-%cyclohexane in two steps from 25° C. to 270° C.; decomposition atT >270° C. PP415-P35 suspended 158.4 mg of PP415-P1 in 0.2 mL ofFT-Raman: Raman: corresponds to class 4, MeCN/H₂O obtained two clear,separated phases; PP415P35.0 contains solvent signals equilibratedsolution at 24° C. shaking at 400 rpm; after PXRD: 326a PXRD:corresponds to class 4 3 d observed thick suspension; added 0.1 mL ofthe TG-FTIR: a4363 TG-FTIR: loss of 2.9 wt.-% MeCN solvent mixture;continued equilibration of suspension from 25° C. to 250° C.;decomposition at 24° C. shaking at 400 rpm; after a total of 5 d atT >250° C. recovered solid material by filter centrifugation (0.20-μmPTFE membrane); examined material by FT-Raman spectroscopy; driedmaterial for 10 min under vacuum (10-20 mbar); examined material by PXRDand TG- FTIR. PP415-P36 dried material of PP415-P35 under vacuum at 80°C. and FT-Raman: Raman: corresponds to class 4 <1 × 10⁻³ mbar; after 3 dexamined material by FT- PP415P36.0 PXRD: corresponds to class 4 Ramanspectroscopy, PXRD and TG-FTIR. PXRD: 339a TG-FTIR: loss of ~0.6 wt.-%TG-FTIR: a4369 (probably H₂O and/or MeCN) in two steps from 25° C. to280° C.; decomposition at T >280° C. PP415-P37 dried material ofPP415-P35 under N₂ flow at 80° C.; FT-Raman: Raman: corresponds to class4 after 3 d examined material by FT-Raman spectroscopy, PP415P37.0 PXRD:corresponds to class 4 PXRD, TG-FTIR, DSC, and DVS. PXRD: 340a TG-FTIR:loss of ~0.9 wt.-% TG-FTIR: a4370 (probably H2O and/or MeCN) in two DSC:d_9907 steps from 25° C. to 280° C.; DVS: dvs1176 decomposition atT >280° C. post-DVS PXRD: DSC: sharp endothermic peak at T = 363a 196.1°C. (ΔH = 29.31 J/g); no decomposition up to 270° C. DVS: slightlyhygroscopic; Δm = +0.7% (50%→85% r.h.); total mass gain of 2.1 wt.-%from 0% r.h. to 95% r.h. post-DVS PXRD: corresponds to class 4 PP415-P38DSC experiment: combined 1.275 mg of PP415-P1 and DSC: d_9917 DSC 1.344mg of PP415-P36; equilibrated for 3 min under N₂; heated sample from−50° C. to 270° C. at 10 K/min. PP415-P39 DSC experiment: combined 2.17mg of PP415-P1 and DSC: d_9923 DSC 2.20 mg of PP415-P36; mixed solidsusing a spatula; equilibrated for 3 min under N₂; heated sample from−50° C. to 173° C. at 10 K/min; held at 173° C. for 30 min; heated from173° C. to 270° C. at 10 K/min. PP415-P40 received ~5 g of 63415, batch#: 2083-69-DC on May PXRD: 390a PXRD: corresponds to class 2 27, 2011;MW = 554.7 g/mol, C₃₃H₄₄F₂N₂O₃ PP415-P41 suspended 101.3 mg of PP415-P40in 0.20 mL of PXRD: 400a PXRD: corresponds to class 5 THF/H₂O (1:1);obtained white suspension; equilibrated suspension at 24° C.; after 3days recovered solid by filter centrifugation (0.2-μm PTFE membrane);dried solid material under vacuum for 5 min; examined solid by PXRD.PP415-P42 dissolved 104.6 mg of PP415-P40 in 0.20 mL of THF; PXRD: 405aPXRD: amorphous (class 1) obtained clear solution; evaporated solventunder N₂ flow overnight; obtained white solid; examined solid by PXRD.PP415-P43 dissolved 101.8 mg of PP415-P40 in 0.20 mL of PXRD: 429a PXRD:corresponds to class 2 THF/hexane (8:2); obtained clear solution;evaporated solvent under N2 flow overnight; obtained white solid;examined solid by PXRD. PP415-P44 dried material of PP415-P41 undervacuum (~100 mbar) PXRD: 474a, PXRD: both mainly amorphous (class at 80°C.; examined solid after 1 day by PXRD (474a); 482a 1), some broad peakswith low continued drying overnight; examined solid again by TG-FTIR:a4401 intensity PXRD (482a) and TG-FTIR. TG-FTIR: ~0.9 wt.-% THF 25-280°C., decomposition at T >300° C. PP415-P45 suspended 3.03 g of PP415-P40in 6.0 mL of THF/H2O PXRD: 471a PXRD: corresponds to class 5 (1:1);obtained white suspension; equilibrated suspension at r.t.; after 1 dayrecovered small aliquot by filter centrifugation and examined it byPXRD; recovered solid of whole sample by vacuum filtration; dried samplefor 10 min under vacuum (~10 mbar). PP415-P46 dried material ofPP415-P45 under vacuum (~100 mbar) PXRD: 481a, PXRD: both mainlyamorphous (class at 80° C.; examined solid after drying overnight by496a 1), some broad peaks with low PXRD (481a); continued drying; after4 days examine TG-FTIR: a4410 intensity solid as PP415-P46a by PXRD(496a) and TG-FTIR. TG-FTIR: ~0.4 wt.-% H₂O 25-250° C., decomposition atT >250° C. PP415-P47 dissolved 101.8 mg of PP415-P40 in 0.4 mL of EtOAc;PXRD: 492a PXRD: corresponds to class 2 obtained clear solution;evaporated solvent under N₂ TG-FTIR: a4412 TG-FTIR: ~6.2 wt.-% EtOAc25-170° C., flow overnight; obtained white solid; examined solid by 1.7wt.-% EtOAc 170-240° C., PXRD and TG-FTIR. decomposition at T >240° C.PP415-P48 dissolved 101.1 mg of PP415-P40 in 0.4 mL of ethyl PXRD: 493aPXRD: corresponds to class 2 formate; obtained clear solution;evaporated solvent TG-FTIR: a4413 TG-FTIR: ~3.5 wt.-% ethyl formateunder N₂ flow overnight; obtained white solid; 25-200° C., decompositionat T >200° C. examined solid by PXRD and TG-FTIR. PP415-P49 dissolved205.3 mg of PP415-P40 in 0.3 mL of acetone; PXRD: 593a PXRD: amorphous(class 1) obtained clear solution; added dropwise to 30.0 mL of H₂O(pre-cooled to 5° C.); obtained thin white suspension; stirred thinsuspension at 5° C. overnight; obtained thicker white suspension;recovered solid by vacuum filtration (pore size P4); obtained 188.3 mgof white solid; examined solid by PXRD.

TABLE 31 Parameters of FIG. 51 NOx Levels (% vs LPS Controls) Compound13 mg/kg 25 mg/kg 50 mg/kg RTA 405 * 44% 26% 18% 63415 30% 18% 16%

TABLE 32 63415: Primary In Vivo ADMET - Key Primary ADMET Assays andEndpoints Assay Key Endpoints 14-day mouse toxicity Tolerability, bodyweight, clinical chemistry Tissue distribution Nrf2 target gene mRNAexpression & enzyme activation in liver 14-day rat toxicityTolerability, body weight, clinical chemistry, & limited histopathologyTissue distribution and plasma TK Nrf2 target gene mRNA expression &enzyme activation in liver 14-day monkey toxicity Tolerability, bodyweight, clinical chemistry, & limited histopathology Tissue distributionand plasma TK Nrf2 target gene mRNA expression and enzyme activation inmultiple tissues & PBMCs

TABLE 33 Parameters of FIG. 54 Vehicle 63415 Dose (mg/kg) 0 10 30 100ALT (U/L) 100 39 63 91 AST (U/L) 156 98 147 167 ALP (U/L) 120 131 110 98Tot Bil (mg/dL) <0.2 <0.2 <0.2 <0.2 BUN (mg/dL) 17 15 15 15 Cr (mg/dL)<0.2 <0.2 <0.2 <0.2 Glu (mg/dL) 288 307 285 273

TABLE 34 63415 is Negative for Genotoxicity in the In Vivo MicronucleusStudy Change Number of PCE/Total from Number of MPCE/ TreatmentErythrocytes Control MPCE/1000 PCE PCE (n = 5/group) (Mean +/− SD) (%)(Mean +/− SD) Scored 24-h timepoint Sesame Oil 0.588 ± 0.04 — 0.2 ± 0.272/10000  125 mg/kg 0.543 ± 0.03 −8 0.3 ± 0.27 3/10000  250 mg/kg 0.520 ±0.06 −12 0.3 ± 0.27 3/10000  500 mg/kg 0.426 ± 0.07 −28 0.0 ± 0.000/10000 1000 mg/kg 0.498 ± 0.05 −15 0.2 ± 0.27 2/10000 1500 mg/kg 0.499± 0.06 −15 0.4 ± 0.22 4/10000 2000 mg/kg 0.531 ± 0.05 −10 0.2 ± 0.272/10000 48-h timepoint Sesame Oil 0.526 ± 0.05 — 0.3 ± 0.27 3/10000  125mg/kg 0.453 ± 0.03 −14 0.2 ± 0.27 2/10000  250 mg/kg 0.391 ± 0.02 −260.2 ± 0.27 2/10000  500 mg/kg 0.339 ± 0.05 −36 0.3 ± 0.45 3/10000 1000mg/kg 0.344 ± 0.04 −35 0.1 ± 0.22 1/10000 1500 mg/kg 0.376 ± 0.05 −390.4 ± 0.42 4/10000 2000 mg/kg 0.360 ± 0.03 −32 0.1 ± 0.22 1/10000

TABLE 35 Parameters of FIG. 35 ALT AST ALP Tot Bil BUN Cr Tot ProtAlbumin Glucose Chol TG Treatment Day (U/L) (U/L) (U/L) (mg/dL) (mg/dL)(mg/dL) (g/dL) (g/dL) (mg/dL) (mg/dL) (mg/dL) Vehicle BL 30 29 320 0.1523 0.63 7.2 4.1 87 124 52 Day 14 37 37 345 0.23 18 0.63 6.9 4.1 63 13064  10 mg/kg BL 46 32 351 0.18 35 0.78 7.4 4 74 146 51 Day 14 46 38 3820.23 27 0.68 7.2 4 39 144 82  30 mg/kg BL 32 32 409 0.18 23 0.7 7.3 4.285 125 47 Day 14 47 43 416 0.2 20 0.58 7.2 4 53 122 64 100 mg/kg BL 3235 381 0.15 24 0.7 6.9 4 96 137 37 Day 14 43 37 390 0.18 24 0.55 6 3.232 93 61

TABLE 36 In Vitro Activity of 63415 and 63355 63415 63355 NO IC₅₀ (nM),RAW264.7 4.0 ± 1   0.63 ± 0.06 WST-1 IC₅₀ (nM), RAW264.7 125 150NQO1-ARE (fold at 62.5 nM in 5.3 ± 1.0 6.5 ± 0.9 HuH7)

TABLE 37 Parameters of FIG. 52 Compound Plasma Whole Blood Brain LiverLung Kidney RTA 405 (nM) 130 1165 93 1143 1631 2357 63415 (nM) 51 6791081 985 533 1604

TABLE 38 Parameters of FIG. 53 Compound Liver Lung Kidney RTA 405 1.931.48 8.25 63415 10.9 1.75 10.9

All of the compounds, polymorphs, formulations, and methods disclosedand claimed herein can be made and executed without undueexperimentation in light of the present disclosure. While the compounds,polymorphs, formulations, and methods of this invention have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompounds, polymorphs, formulations, and methods, as well as in thesteps or in the sequence of steps of the method described herein withoutdeparting from the concept, spirit, and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A compound of the formula:

or a pharmaceutically acceptable salt thereof. 2-3. (canceled)
 4. Apolymorphic form of a compound having the formula:

wherein the polymorphic form has an X-ray powder diffraction pattern(CuKα) comprising a halo peak at about 14°2θ.
 5. The polymorphic form ofclaim 4, wherein the X-ray powder diffraction pattern (CuKα) furthercomprises a shoulder peak at about 820°2θ.
 6. The polymorphic form ofclaim 4, wherein the X-ray powder diffraction pattern (CuKα) issubstantially as shown in FIG.
 59. 7. The polymorphic form of claim 4,further having a T_(g) from about 150° C. to about 155° C. 8-9.(canceled)
 10. The polymorphic form of claim 4, further having adifferential scanning calorimetry (DSC) curve comprising an endothermcentered from about 150° C. to about 155° C. 11-12. (canceled)
 13. Thepolymorphic form of claim 4, having a differential scanning calorimetry(DSC) curve substantially as shown in FIG.
 62. 14. A polymorphic form ofa compound having the formula:

wherein the polymorphic form is a solvate having an X-ray powderdiffraction pattern (CuKα) comprising significant peaks at about 5.6,7.0, 10.6, 12.7, and 14.6°2θ.
 15. The polymorphic form of claim 14,wherein the X-ray powder diffraction pattern (CuKα) is substantially asshown in FIG. 75, top pattern.
 16. A polymorphic form of a compoundhaving the formula:

wherein the polymorphic form is a solvate having an X-ray powderdiffraction pattern (CuKα) comprising significant peaks at about 7.0,7.8, 8.6, 11.9, 13.9 (double peak), 14.2, and 16.0 °2 θ.
 17. Thepolymorphic form of claim 16, wherein the X-ray powder diffractionpattern (CuKα) is substantially as shown in FIG. 75, second pattern fromtop.
 18. A polymorphic form of a compound having the formula:

wherein the polymorphic form is an acetonitrile hemisolvate having anX-ray powder diffraction pattern (CuKα) comprising significant peaks atabout 7.5, 11.4, 15.6, and 16.6 °2θ.
 19. The polymorphic form of claim18, wherein the X-ray powder diffraction pattern (CuKα) is substantiallyas shown in FIG. 75, second pattern from bottom.
 20. The polymorphicform of claim 18, further having a T_(g) of about 196° C.
 21. Thepolymorphic form of claim 18, further having a differential scanningcalorimetry (DSC) curve comprising an endotherm centered at about 196°C.
 22. The polymorphic form of claim 18, having a differential scanningcalorimetry (DSC) curve substantially as shown in FIG.
 116. 23. Apolymorphic form of a compound having the formula:

wherein the polymorphic form is a solvate having an X-ray powderdiffraction pattern (CuKα) comprising significant peaks at about 6.8,9.3, 9.5, 10.5, 13.6, and 15.6 °2θ.
 24. The polymorphic form of claim23, wherein the X-ray powder diffraction pattern (CuKα) is substantiallyas shown in FIG. 75, bottom pattern.
 25. A pharmaceutical compositioncomprising: an active ingredient consisting of a compound of claim 1,and a pharmaceutically acceptable carrier. 26-42. (canceled)
 43. Amethod of treating or preventing a condition associated withinflammation or oxidative stress in a patient in need thereof,comprising administering to the patient a therapeutically effectiveamount of the pharmaceutical composition of claim
 25. 44. The method ofclaim 43, wherein the condition is associated with inflammation.
 45. Themethod of claim 43, wherein the condition is associated with oxidativestress.
 46. The method of claim 43, wherein the condition is a skindisease or disorder, sepsis, dermatitis, osteoarthritis, cancer,inflammation, an autoimmune disease, inflammatory bowel disease, acomplication from localized or total-body exposure to ionizingradiation, mucositis, acute or chronic organ failure, liver disease,pancreatitis, an eye disorder, a lung disease, or diabetes. 47-95.(canceled)